Toxin conjugates

ABSTRACT

The present invention relates to a toxin conjugate in which a residue derived from a compound having an affinity for a target cell is bound to a toxin through a spacer comprising polyalkylene glycol and dipeptide.

This application is a 371 of PCT/JP96/01241, filed on May 10, 1996.

TECHNICAL FIELD

The present invention relates to toxin conjugates in which a toxin isbound through a spacer to a residue derived from a compound which has anaffinity for a target cell, for example, a residue derived from anantibody or antibody fragment which is specific to a cancer. The toxinconjugate obtained by the present invention inhibits the growth of atarget cell selectively and efficiently, and is useful as an activeingredient of an antitumor agent.

BACKGROUND ART

Anthracycline anticancer compounds so far known include daunomycin (U.S.Pat. No. 3,590,028) and adriamycin (U.S. Pat. No. 3,590,028), which arein wide clinical use as anticancer agents. However, side effects ofthese compounds have been reported; for example, adriamycin is known tohave side effects such as cardial toxicity and marrow depression [CancerChemotherapy and Pharmacology, 4, 5-10 (1980)]. Alleviation of such sideeffects is a big problem to be solved, and comprehensive research has sofar been made to this end. Specifically, in recent years, research ondrug delivery systems has been pursued aiming at alleviation oftoxicity, maintenance of concentration in blood and improvement ofaffinity for a cancer cell. For example, the modification with acopolymer of divinyl ether-maleic anhydride (Japanese PublishedUnexamined Patent Application No. 67490/85), and the modification withdextran [Cancer Treatment Reports, 66, 107 (1982)] have been reported.

Further, antibody conjugates (toxin conjugates) having a specificity toa cancer cell have been studied. Some examples of such conjugates areshown below [Bioconjugate Chem., 1, 13 (1990)].

    __________________________________________________________________________    stracture                      toxin                                          __________________________________________________________________________     ##STR1##                      vinblastine                                     ##STR2##                      risin A  diphtheria toxin A  abrin A            ##STR3##                      vinblastine hydrazide  methotrexate                                           hydrazide                                       ##STR4##                      anthracycline                                   ##STR5##                      chelates of indium  and yttrium                 ##STR6##                      metal chelates                                  ##STR7##                      anthracycline                                  __________________________________________________________________________

There are some other reports relating to antibody conjugates [JapanesePublished Unexamined Patent Application No. 67433/85; Japanese PublishedUnexamined Patent Application No. 35575/88; Japanese PublishedUnexamined Patent Application No. 150282/88; Japanese PublishedUnexamined Patent Application No. 246336/88; Biochem. J., 173, 723(1978); Cancer Res., 50, 6600 (1990); Science, 261, 212 (1993);Bioconjugate Chem., 4, 275 (1993); Bioconjugate Chem., 4, 251 (1993);Bioconjugate Chem., 5, 88 (1994); Bioconjugate Chem., 5, 31 (1994); andBioconjugate Chem., 5, 246 (1994)].

There are also known examples in which low molecular weight polyethyleneglycol is used as a spacer [Proc. Natl. Acad. Sci. USA, 88, 9287 (1991);PCT National Publication No. 508856/93; and Bioconjugate Chem., 4, 455(1993)], and examples of the modification of an antibody withpolyethylene glycol (WO 93/08838 and WO 86/04145). Further, the use of aspacer containing a peptide has been reported [U.S. Pat. No. 4,671,958;PCT National Publication No. 502886/93; and Bioconjugate Chem., 4, 10(1993)].

DISCLOSURE OF THE INVENTION

The present inventors made intensive studies in search of an excellenttoxin conjugate which kills tumor cells selectively. As a result, theinventors have found that a conjugate having a spacer which isspecifically cleaved when introduced into a specific cell can beobtained by chemically binding a toxin to a compound which has aspecific affinity for a cancer cell through a novel spacer comprisingpolyethylene glycol and dipeptide. Thus the present invention has beencompleted.

The present invention relates to a toxin conjugate in which a residuederived from a compound having an affinity for a target cell is bound toa toxin through a spacer comprising polyalkylene glycol and dipeptide.

Typical examples of the conjugates of the present invention are toxinconjugates represented by general formula (A):

    --Z.paren open-st.X.sup.0 --(OAlk).sub.n O--W.sup.0 --R.sup.1 --R.sup.2 --W.sup.1 --Y).sub.m                                      (A)

wherein Z represents a residue derived from a compound having anaffinity for a target cell; Y¹ represents a toxin; R¹ and R², which maybe the same or different, each represents an amino acid residue; Alkrepresents alkylene; n represents an integer of 1-1000; and m representsan integer of 1-100. Although X⁰, W⁰ and W¹ are not specificallydefined, examples of their representations are as follows: X⁰ represents--COAlk¹ --, --SAlk¹ --, --COOAlk¹ --, --CONHAlk¹ --, --COAlk¹ CO--,##STR8## W⁰ represents CO, --Alk¹ CO--, or --Alk¹ S--; and W¹ representsa single bond, S, --OAlk¹ CO--, --NHAlk¹ CO--, --NHAlk¹ NH--, ##STR9##In the above formulae, Alk¹ and Alk², which may be the same ordifferent, each represents a straight-chain or branched alkylene having1-8 carbon atoms, such as methylene, ethylene, propylene, isopropylene,butylene, isobutylene, pentylene, hexylene, heptylene, and octylene.

Particularly, preferred toxin conjugates are compounds represented bygeneral formula (I):

    Z.paren open-st.X.sup.1 --CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n OCH.sub.2 CO--R.sup.1 --R.sup.2 --W--Y.sup.1).sub.m                 (I)

wherein X¹ represents CO, S or ##STR10## w represents a single bond or##STR11## and Z, Y¹, R¹, R², n and m have the same meanings as definedabove. The compounds represented by general formula (I) are hereinafterreferred to as Compounds (I), and the same applies to the compounds ofother formula numbers.

In the definitions of the above-described groups, the alkylene moiety ofthe alkylene and the polyalkylene glycol means a straight-chain orbranched alkylene having 1-8 carbon atoms such as methylene, ethylene,propylene, isopropylene, butylene, isobutylene, pentylene, hexylene,heptylene, and octylene. Examples of the compounds which have anaffinity for a target cell are compounds having a structure capable ofbinding to X¹ such as COOH, NH, SH, and OH, e.g., receptor ligands suchas epidermal growth factors (EGF) and transferrin having an affinity fora target cell, adhesion molecules represented by thearginine-glycine-aspartic acid sequence, and proteins and peptides suchas antibodies and antibody fragments. Preferred examples are antibodiesand antibody fragments. The antibodies include polyclonal antibodies andmonoclonal antibodies produced according to known methods which belongto immunoglobulin (Ig) classes such as IgG, IgA, IgM, and IgE, andimmunoglobulin subclasses, for example, IgG₁, IgG₂, IgG₃, and IgG₄ inthe case of IgG. Preferred examples are KM-641 antibody which is anantibody against ganglioside GD₃ which is highly expressed in a cancercell (Japanese Published Unexamined Patent Application No. 176791/93),KM-231 (AMC-462) antibody which is an antibody against sialyl Lewis a(Japanese Published Unexamined Patent Application No. 021562/88), andNL-1 antibody which is an antibody against common human acute lymphaticleukemia cell antigen (CALLA) [Proc. Natl. Acad. Sci. USA 79, 4386-4390(1982)]. Examples of the antibody fragments are F(ab')₂ obtained bytreating the above-mentioned antibodies with a proteolytic enzyme suchas pepsin, Fab' obtained by reducing F(ab')₂ with mercaptan, and Fabobtained by degrading the antibodies with a proteolytic enzyme such aspapain, trypsin, chymotrypsin, and plasmin. F(ab')₂, Fab', and Fab areknown as well as methods for producing them [Immunochemistry, YuichiYamamura et al., p. 461, Asakura Shoten (1973)]. Examples of the toxinsare toxins having a structure capable of condensing with a carboxylgroup of the terminal amino acid R² or capable of attaching to a doublebond of maleinimide, such as NH, SH and OH, e.g., anthracyclinecompounds such as adriamycin (U.S. Pat. No. 3,590,028) and daunorubicin(U.S. Pat. No. 3,616,242), duocarmycin derivatives such as DC-88Aderivatives (Japanese Published Unexamined Patent Application No.288879/90) and the compounds described in Reference Examples, mitomycinA, mitomycin C, and protein toxins such as ricin A, diphtheria toxin,and Pseudomonas exotoxin. Examples of the amino acid residues are analanine residue, a leucine residue, a glycine residue, a proline residueand a valine residue.

The abbreviations used herein have the following meanings, unlessotherwise specified.

The abbreviations for amino acids and their protecting groups follow therecommendations by IUPAC-IUB Joint Commission on BiochemicalNomenclature [Biochemistry, 11, 1726 (1972)].

Ala: L-Alanine

Val: L-Valine

Pro: L-Proline

Gly: Glycine

DMF: N,N-Dimethylformamide

DMSO: Dimethylsulfoxide

THF: Tetrahydrofuran

TFA: Trifluoroacetic acid

NMM: N-Methylmorpholine

Bzl: Benzyl

tBu: tert-Butyl

Z: Benzyloxycarbonyl

Pic: Picolyl

HONSu: N-Hydroxysuccinimide

ONSu: Succinimidoxy

DCC: N,N'-Dicyclohexylcarbodiimide

DCU: N,N'-Dicyclohexylurea

ADM: Adriamycin

DNR: Daunorubicin

HOBt: N-Hydroxybenzotriazole

PyBOP: Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

DMAP: 4-(N,N-Dimethylamino) pyridine

PEG: COCH₂ (OCH₂ CH₂)_(n) OCH₂ CO

HPLC: High performance liquid chromatography

NMR: Nuclear magnetic resonance

The processes for preparing Compounds (I) and polyethylene glycolderivatives represented by general formula (II):

    X.sup.2 --CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n OCH.sub.2 CO--R.sup.1 --R.sup.2 --Y.sup.2                                       (II)

(wherein X² represents carboxyl, mercapto or ##STR12## Y² representshydroxyl or ##STR13## and R¹, R² and n have the same meanings as definedabove) are described below.

Process 1: Process for preparing Compound (Ia), i.e., Compound (I)wherein Z is a group having N, S or O, X¹ is CO, and W is a single bond

Compound (Ia) can be prepared according to the following reaction steps.##STR14## (In the formulae, A¹ and A², which may be the same ordifferent, each represents a carboxylic acid protecting group; A³ andA⁴, which may be the same or different, each represents a carbxylic acidactivating group; Hal represents halogen; Z¹ represents a group havingN, S or O in the definition of Z; and Y¹, R¹, R² and n have the samemeanings as defined above.)

Examples of the carboxylic acid protecting group are carboxylic acidprotecting groups used in ordinary peptide synthesis (Fundamentals andExperiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen) such astBu, Bzl, and Pic. An example of the carboxylic acid activating group isONSu. The halogen means a chlorine atom, a bromine atom or an iodineatom.

(Step 1)

Compound (V) can be obtained by reaction of polyethylene glycoldicarboxylic acid (III) with Compound (IV) in an amount of 0.1 to 1equivalent, preferably, 0.5 equivalent in a solvent such as DMF in thepresence of a base such as potassium carbonate at -50 to 30° C. for 1 to24 hours. Diester and unreactive dicarboxylic acid contained in theobtained product can be removed by partition column chromatography,column chromatography using adsorption resins, reversed-phase silicagel, alumina, diatomaceous earth, or ion-exchange resins, preferably,silica gel column chromatography or thin layer chromatography.

(Step 2)

Compound (VII) can be obtained by condensing Compound (V) with Compound(VI) obtained according to an ordinary liquid-phase peptide synthesismethod (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiyaet al., Maruzen) in a solvent in the presence of a base in an amount of1 to 2 equivalents, using a condensing agent in an amount of 1 to 10equivalents, preferably 1 to 2 equivalents. Examples of the base aretriethylamine and NMM, examples of the condensing agent are ordinaryamino acid condensing reagents such as DCC and EDC, and examples of thesolvent are methylene chloride, chloroform and DMF. The reaction iscarried out by stirring at 0 to 30° C. for 1 to 24 hours.

It is preferred that A² used as the carboxylic acid protecting group ofCompound (VI) is a group which can be selectively removed separatelyfrom A¹ of Compound (V).

Compound (VII) can also be obtained by condensing Compound (V) withHONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably1 to 2 equivalents in a solvent in the presence of an equivalent amountof a base, using a condensing agent in an amount of 1 to 5 equivalents,preferably 1 to 2 equivalents to obtain an active ester, and then bysubjecting the obtained ester to reaction with Compound (VI) at 0 to 30°C. for 1 to 24 hours. As the base, condensing agent and solvent, thosewhich are described above can be used.

(Step 3)

Compound (VIII) can be obtained by selectively removing the protectinggroup A² from Compound (VII) according to a method for the removal of aprotecting group used in ordinary peptide synthesis (Fundamentals andExperiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).

(Step 4)

Compound (X) can be obtained by condensing Compound (VIII) with anequivalent amount of a toxin in a solvent in the presence of a base inan amount of 1 to 2 equivalents, using a condensing agent in an amountof 1 to 10 equivalents, preferably 1 to 2 equivalents. Examples of thebase are triethylamine and NMM, examples of the condensing agent areordinary amino acid condensing reagents such as DCC and EDC, andexamples of the solvent are methylene chloride, chloroform and DMF. Thereaction is carried out by stirring at -30 to 30° C. for 1 to 24 hours.

Compound (X) can also be obtained by condensing Compound (VIII) withHONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably1 to 2 equivalents in a solvent in the presence of an equivalent amountof a base, using a condensing agent in an amount of 1 to 5 equivalents,preferably 1 to 2 equivalents to obtain an active ester (IX), and thenby subjecting the obtained ester to reaction with a toxin at -30 to 30°C. for 1 to 24 hours. As the base, condensing agent and solvent, thosewhich are described above can be used.

(Step 5)

Compound (XI) can be obtained by removing the protecting group A¹ fromCompound (X) according to a method for the removal of a protecting groupused in ordinary peptide synthesis (Fundamentals and Experiments ofPeptide Synthesis, Nobuo Izumiya et al., Maruzen). In the case, forexample, where Bzl is used as A¹ and tBu as A², which are to be removedin Steps 3 and 5, respectively, deprotection is carried out according toordinary methods for selectively removing amino acid protecting groupssuch as hydration in the presence of a palladium carbon catalyst for A¹,and trifluoroacetic acid treatment for A², whereby A¹ and A² can beselectively removed in each step. It is possible to use the abovecombination of A¹ and A² in reverse and to reverse the order ofdeprotection steps.

Compound (XI) can also be obtained by removing the protecting group A¹from Compound (IX) obtained in Step 4 according to the method of Step 5,and then subjecting the obtained compound to reaction with a toxinaccording to the method of Step 2.

(Step 6)

Compound (Ia) can be obtained from Compound (XI) and a compound whichhas an affinity for a target cell and has NH, SH, or OH in the moleculeaccording to the method of Step 2. The compounds having an affinity fora target cell, such as proteins and peptides, are liable to be denaturedand inactivated in an organic solvent, and it is preferred to carry outthe above reaction under mild conditions, e.g. in an aqueous solution.In this case, the reaction is carried out by dissolving a compoundhaving an affinity for a target cell in a buffer such as a phosphatebuffer or a borate buffer (pH 6-8), and adding to the solution Compound(XI) in an amount of 1 to 500 equivalents, preferably, 1 to 50equivalents, and a condensing agent such as EDC, followed by stirring at0 to 30° C. for 1 to 48 hours. Alternatively, the reaction may becarried out by obtaining an active ester (XII) according to the methodof Step 2, and adding to a solution of a compound having an affinity fora target cell in a buffer (pH 6-8) the obtained active ester in anamount of 1 to 500 equivalents, preferably 1 to 50 equivalents in thepresence of 0 to 10%, preferably 0 to 5% DMSO or DMF, followed bystirring at 0 to 30° C. for 1 to 48 hours.

Process 2: Process for preparing Compound (Ib), i.e., Compound (I)wherein Z is a group having N, S or O, X¹ is CO, and W is ##STR15##

Compound (Ib) can be prepared according to the following reaction steps.##STR16## (In the formulae, A¹, Y¹, Z¹, R¹, R² and n have the samemeanings as defined above).

(Step 7)

Compound (XIII) can be obtained from Compound (VIII) and aminoethylmaleimide according to the method of Step 2.

(Step 8)

Compound (XIV) can be obtained by subjecting Compound (XIII) to reactionwith a toxin. The reaction is carried out by dissolving a toxin in abuffer such as a phosphate buffer and a borate buffer (pH 6-8), andadding Compound (XIII) in an amount of 1 to 50 equivalents to thesolution, followed by stirring at 0 to 30° C. for 1 to 48 hours.

(Step 9)

Compound (Ib) can be obtained from Compound (XIV) according to themethods of Steps 5 and 6.

Process 3: Process for preparing Compound (Ic), i.e., Compound (I)wherein Z is a group having CO, X¹ is S, and W is a single bond

Compound (Ic) can be prepared according to the following reaction steps.##STR17## (In the formulae, A⁴ represents a thiol protecting group; Z²represents a group having CO in the definition of Z; and A¹, Y¹, R¹, R²and n have the same meanings as defined above.)

Examples of the thiol protecting group are benzyl, picolyl, andnitrobenzyl.

The starting compound (XV) can be obtained according to the method forthe synthesis of polyethylene glycol derivatives described in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications(J. M. Harris, Ed., Plenum, N.Y. 1992).

(Step 10)

Compound (XVI) can be obtained by protecting the thiol group of Compound(XV) according to a method for introducing a protecting group used inordinary peptide synthesis (Fundamentals and Experiments of PeptideSynthesis, Nobuo Izumiya et al., Maruzen).

(Step 11)

Compound (XVII) can be obtained from Compound (XVI) according to themethod of Step 3.

(Step 12)

Compound (XVIII) can be obtained from Compound (XVII) according to themethods of Steps 2 and 3.

(Step 13)

Compound (XIX) can be obtained from Compound (XVIII) according to themethod of Step 4.

(Step 14)

Compound (XX) can be obtained by deprotecting Compound (XIX) accordingto a method for removing a protecting group used in ordinary peptidesynthesis (Fundamentals and Experiments of Peptide Synthesis, NobuoIzumiya et al., Maruzen).

(Step 15)

Compound (Ic) can be obtained by binding Compound (XX) to a compoundhaving an affinity for a target cell and having COOH in the molecule bya method such as the activation of a thiol group described in J. AppliedBiochem., 6, 56-63 (1984).

Process 4: Process for preparing Compound (Id), i.e., Compound (I)wherein Z is a group having N, S or O, X¹ is ##STR18## and W is a singlebond, and Compound (IId), i.e., Compound (II) wherein X² is ##STR19##and Y² is hydroxyl.

Compound (Id) and Compound (IId) can be prepared according to thefollowing reaction steps. ##STR20## (In the formulae, A¹, Y¹, Z¹, R¹, R²and n have the same meanings as defined above.)

The starting compound (XXI) can be obtained according to the method forthe synthesis of polyethylene glycol derivatives described in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications(J. M. Harris, Ed., Plenum, N.Y. 1992).

(Step 16)

Compound (XXII) can be obtained from Compound (XXI) according to themethod of Step 3.

(Step 17)

Compound (IId) can be obtained from Compound (XXII) according to themethods of Steps 2 and 3.

(Step 18)

Compound (XXIII) can be obtained from Compound (IId) according to themethod of Step 4.

(Step 19)

Compound (Id) can be obtained from Compound (XXIII) and a compoundhaving an affinity for a target cell and having NH, SH or OH in themolecule according to the method of Step 8.

Process 5: Process for preparing Compound (IIa), i.e., Compound (II)wherein X² is carboxyl and Y² is hydroxyl

(Step 20)

Compound (IIa) can be obtained from Compound (VIII) according to themethod of Step 3.

Process 6: Process for preparing Compound (IIb), i.e., Compound (II)wherein X² is carboxyl and Y² is ##STR21## (Step 21)

Compound (IIb) can be obtained from Compound (XIII) according to themethod of Step 3.

Process 7: Process for preparing Compound (IIc), i.e., Compound (II)wherein X² is mercapto and Y² is hydroxyl

(Step 22)

Compound (IIc) can be obtained from Compound (XVIII) according to themethod of Step 14.

Compounds (I) and (II) having the desired groups at the desiredpositions can be obtained by combining the above-described methodsappropriately.

The intermediates and desired compounds in the above-described processescan be isolated and purified by purification methods such as filtration,extraction, washing, drying, concentration, recrystallization, variouskinds of column chromatography, e.g. silica gel chromatography,ion-exchange chromatography, reversed-phase chromatography, and gelfiltration chromatography, and dialysis using an ordinary semipermeablemembrane. The intermediates can be subjected to the subsequent reactionwithout a specific purification treatment.

Examples of Toxin Conjugates (I) obtained by the above-describedprocesses are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Compound No.                                                                              Structure                                                         ______________________________________                                        Ia-1        NL-1-(PEG-Ala-Val-ADM).sub.m                                      Ia-2        NL-1-(PEG-Ala-Pro-ADM).sub.m                                      Ia-3        NL-1-(PEG-Gly-Pro-ADM).sub.m                                      Ia-4        KM-231-(PEG-Ala-Val-DNR).sub.m                                    Ia-5        KM-231-(PEG-Ala-Pro-DNR).sub.m                                    Ia-6        KM-231-(PEG-Gly-Pro-DNR).sub.m                                    Ia-7        NL-1-[PEG-Ala-Val-Compound (20) *].sub.m                          Ia-8        NL-1-[PEG-Ala-Pro-Compound (20) *].sub.m                          Ia-9        NL-1-[PEG-Gly-Pro-Compound (20) *].sub.m                          Ia-10       KM-231-[PEG-Ala-Val-Compound (12) **].sub.m                       Ia-11       KM-641-[PEG-Ala-Val-Compound (12) **].sub.m                       ______________________________________                                         *: Synthesized in Reference Example 17                                        **: Synthesized in Reference Example 15                                  

The pharmacological activities of the toxin conjugates are shown belowby Test Examples.

TEST EXAMPLE 1

The inhibitory effect of the toxin conjugates on cell growth wasexamined. Cervical cancer HeLaS3 cells (CALLA⁻) having no expression ofCALLA antigen and Burkitt lymphoma Daudi cells (CALLA⁺) having anexpression of CALLA antigen were used as target cell lines. Each of thetarget cell suspensions was put into wells of a 96-well flat plate in anamount of 50 μl (1×10³ cells/well), and cultured in a CO₂ -incubator at37° C. for 2 hours. After culturing, various dilutions of a toxinconjugate or a monoclonal antibody were respectively added in an amountof 50 μl, followed by further culturing in the CO₂ -incubator at 37° C.for 68 hours. Then, 20 μl of ³ H-thymidine (463 KBq/ml) was added toeach well, and after 4 hours, the cells were harvested to determine theradioactivity of ³ H-thymidine incorporated into the cells by usingMatrix 96 (Packard Japan). The cell growth inhibiting activity wascalculated according to the following equation. ##EQU1##

As a result, Compound (Ia-3) and Compound (Ia-1) exhibited a littleinhibitory effect on the growth of HeLaS3 cells at high concentrations,whereas they exhibited a remarkable inhibitory effect on the growth ofDaudi cells even at very low concentrations. When Compound (Ia-9) orCompound (Ia-7) was added, an inhibitory effect was hardly observed onthe growth of HeLaS3 cells, while a more specific inhibitory effect wasobserved on the growth of Daudi cells. Addition of the monoclonalantibody (NL-1) alone had little effect on the cell growth (refer toFIG. 1).

TEST EXAMPLE 2

The inhibitory effect of Compound (Ia-6) on cell growth was examined inthe same manner as in Test Example 1. Cervical cancer HeLaS3 cells(sLe^(a-)) having no expression of sLe^(a) antigen and large intestinecancer SW1116 cells (sLe^(a+)) having an expression of sLe^(a) antigenwere used as target cell lines. As a result, an inhibitory effect wasobserved on the growth of SW1116 cells, but not on the growth of HeLaS3cells. When a monoclonal antibody (KM-231) alone was added, no cellgrowth inhibiting effect was observed on either of these strains. Theantigen-specific cell growth inhibiting effect of the conjugate was thusconfirmed (refer to Table 2).

                  TABLE 2                                                         ______________________________________                                        Compound       Growth inhibiting effect (%)                                   (12.5 μg/ml)                                                                              HeLaS3    SW1116                                               ______________________________________                                        Compound (Ia-6)                                                                              1.6       26.8                                                 KM-231         0.0       0.0                                                  ______________________________________                                    

From the foregoing, the antigen-specific cell growth inhibiting effectof various kinds of conjugates and the utility of the spacers wereconfirmed.

TEST EXAMPLE 3

The inhibitory effect of Compound (Ia-10) on cell growth was examined.Cervical cancer HeLaS3 cells (sLe^(a-)) having no expression of sLe^(a)antigen and large intestine cancer SWI116 cells (sLe^(a+)) having anexpression of sLea antigen were used as target cell lines. Each of thetarget cell suspensions was put into wells of a 96-well flat plate in anamount of 50 μl (1×10³ cells/well), and cultured in a CO₂ -incubator at37° C. for 2 hours. After culturing, various dilutions of thedrug-monoclonal antibody conjugate or a monoclonal antibody wererespectively added in an amount of 50 μl, followed by further culturingin the CO₂ -incubator at 37° C. for 2 hours. Then, the cells in theplate were centrifuged, and immediately after removal of thesupernatant, 100 μl of a medium was added, followed by further culturingfor 64 hours. To each well was added 20 μl of ³ H-thymidine (463KBq/ml), and after 4 hours, the cells were harvested to determine theradioactivity of ³ H-thymidine incorporated into the cells by usingMatrix 96 (Packard Japan). The cell growth inhibiting activity wascalculated according to the following equation. ##EQU2##

As a result, Compound (Ia-10) exhibited an inhibitory effect on thegrowth of SW1116 cells, but no effect on the growth of HeLaS3 cells.When the monoclonal antibody (KM-231) alone was added, an inhibitoryeffect was slightly observed only on the growth of SA1116 cells. Theantigen-specific cell growth inhibiting effect of the conjugate and theutility of the spacer were thus confirmed also in this assay system(refer to Table 3).

                  TABLE 3                                                         ______________________________________                                        Compound        Growth Inhibiting effect (%)                                  (75 μg/ml)   HeLaS3    SW1116                                              ______________________________________                                        Compound (Ia-10)                                                                              0.0       20.8                                                KM-231          0.0       5.3                                                 ______________________________________                                    

TEST EXAMPLE 4

Human myeloma SK-Ly-18 cells were suspended in RPMI-1640 mediumcontaining 10 fetal calf serum at a concentration of 2×10⁸ cells/ml, andthe suspension was mixed with Matrigel (registered trademark; BectonDickinson Labware, USA) in the ratio of 1:1 (v/v). The mixture (0.1 ml,1×10⁷ cells/mouse) was subcutaneously transplanted into BALB/C nu/numice (Clea Japan, Inc.). On the 7th day after the tumor transplantation,a drug-monoclonal antibody conjugate (amount corresponding to 7.5 mg/kgADM) or ADM (7.5 mg/kg) was intravenously administered to the micedivided in groups each consisting of 5. A control group was givenphysiological saline in the same manner. The major axis and the minoraxis of tumor were measured at intervals, and the tumor volume wascalculated as an approximation value of an ellipsoid according to thefollowing equation.

    Tumor Volume (mm.sup.3)=(a×b.sup.2)/2

a: major axis (mm) b: minor axis (mm)

The therapeutic effect on the transplanted tumor was evaluated in termsof V/V₀, the ratio of the tumor volume on the day of evaluation (V) tothat on the day of drug administration (V₀).

As a result, a significant tumor growth was observed in the controlgroup, and a remarkable tumor growth inhibiting effect was observed inCompound (Ia-1)- and Compound (Ia-3)-administered groups. On the otherhand, a growth inhibiting effect was not observed in the test group towhich the same quantity of ADM alone was given compared with the controlgroup. The utility of the drug-monoclonal antibody conjugates was thusdemonstrated (refer to FIG. 2).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cell growth inhibiting effect of toxin conjugates and amonoclonal antibody.

(a) The result obtained using Compound (Ia-3)

(b) The result obtained using Compound (Ia-1)

(c) The result obtained using Compound (Ia-9)

(d) The result obtained using Compound (Ia-7)

(e) The result obtained using NL-1

◯ The result obtained using Daudi cells

 The result obtained using HeLaS3 cells

FIG. 2 shows the therapeutic effect of toxin conjugates and a drug ontransplanted tumor.

 Control group

□ The result obtained using Compound (Ia-1)

Δ The result obtained using Compound (Ia-3)

◯ The result obtained using ADM

BEST MODE FOR CARRYING OUT THE INVENTION

Certain embodiments of the invention are illustrated in the followingExamples and Reference Examples.

EXAMPLE 1

Toxin Conjugate (Ia-1): NL-1-(PEG-Ala-Val-ADM)_(m)

In 500 μl of methylene chloride was dissolved 275 μg (0.21 μmol) ofCompound (XI-1) obtained in Reference Example 6, and 10 μl of a solutionof HONSu (1.1 μmol) in methylene chloride (13.0 mg/ml) and 10 μtl of asolution of DCC (1.1 μmol) in methylene chloride (22.0 mg/ml) were addedsuccessively thereto under ice cooling. After stirring under ice coolingfor 1 hour and then at room temperature for 1.5 hours, the insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. The residue was dissolved in 70 μlof DMSO, and 1400 μl of a phosphate buffer was added thereto. To theresulting mixture was added 480 μl of an aqueous solution of NL-1antibody (3.3 mg/ml), followed by gentle stirring at 4° C. for 24 hours.After the insoluble matter was removed with a filter (0.45 μm), theantibody fraction was purified by gel filtration HPLC [column: Superose12 (Pharmacia Fine Chemicals, Inc.), developer: phosphate buffer, flowrate: 0.5 ml/min, detection: at the absorbance of 280 nm]. The desiredfraction eluted was concentrated using a small-size ultrafiltrationmembrane (Millipore Corp., cut-off molecular weight: 5000) to give 771μg of NL-1-(PEG-Ala-Val-ADM)_(m) (protein content: 0.67 mg/ml) (yield:49%).

In the obtained conjugate, the number of molecules of adriamycin boundwas 2.2 per antibody molecule as calculated from the absorbance at 280nm (absorption of protein=total absorption at 280 nm--absorption ofadriamycin at 280 nm) and that at 495 nm (absorption of adriamycin,ε=1.21×10⁴ M⁻¹ cm⁻¹, ε280=ε495). It was confirmed that the affinity ofthe antibody is approximately equal to that of an unbound antibodyaccording to the fluorescent antibody method described below.

<Measurement of an affinity of an antibody by the fluorescent antibodymethod>

To Daudi cells (1×10⁶) was added the above-described conjugate (10μg/ml), and the mixture was subjected to reaction for 30 minutes underice cooling. The cells were centrifuged and washed with a phosphatebuffer three times, followed by removal of unreactive conjugate. To theresulting mixture was added 20 ml of FITC (fluoresceinisothiocyanate)-labelled anti-mouse IgG antibody (Wako Pure ChemicalIndustries, Ltd., 30 times dilution) and the resulting mixture wassubjected to reaction for 30 minutes under ice cooling. Aftercentrifugation and washing with a phosphate buffer were repeated threetimes, measurement was carried out using a flow cytometer (EPICS Elite,Coulter). As a control, a reaction mixture of cells and theFITC-labelled anti-mouse IgG antibody was used.

EXAMPLE 2

Toxin Conjugate (Ia-2): NL-1-(PEG-Ala-Pro-ADM)_(m)

In 500 μl of methylene chloride was dissolved 289 μg (0.22 μmol) ofCompound (XI-2) obtained in Reference Example 7, and 10 μl of a solutionof HONSu (1.1 μmol) in methylene chloride (13.0 mg/ml) and 10 μl of asolution of DCC (1.1 μmol) in methylene chloride (23.1 mg/ml) were addedsuccessively thereto under ice cooling. After stirring under ice coolingfor 1 hour and then at room temperature for 1.5 hours, the insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. The residue was dissolved in 95 μlof DMSO, and 1900 μl of a phosphate buffer was added thereto. To theresulting mixture was added 510 μl of an aqueous solution of NL-1antibody (3.3 mg/ml), followed by gentle stirring at 4° C. for 24 hours.After the insoluble matter was removed with a filter (0.45 μm), theantibody fraction was purified by gel filtration HPLC and concentratedin the same manner as in Example 1 to give 1060 μg ofNL-1-(PEG-Ala-Pro-ADM)_(m) (protein content: 0.88 mg/ml) (yield: 63%).

In the obtained conjugate, the number of molecules of adriamycin boundwas 1.8 per antibody molecule as calculated from the absorbances at 280nm and 495 nm in the same manner as in Example 1. It was confirmed thatthe affinity of the conjugate was approximately equal to that of anunbound antibody according to the fluorescent antibody method describedin Example 1.

EXAMPLE 3

Toxin Conjugate (Ia-3): NL-1-(PEG-Gly-Pro-ADM)_(m)

In 500 μl of methylene chloride was dissolved 259 μg (0.20 μmol) ofCompound (XI-3) obtained in Reference Example 8, and 10 μl of a solutionof HONSu (1.0 μmol) in methylene chloride (11.7 mg/ml) and 10 μl of asolution of DCC (1.0 μmol) in methylene chloride (21.0 mg/ml) were addedsuccessively thereto under ice cooling. After stirring under ice coolingfor 1 hour and then at room temperature for 1.5 hours, the insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. The residue was dissolved in 76 μlof DMSO, and 1460 μl of a phosphate buffer was added thereto. To theresulting mixture was added 460 μl of an aqueous solution of NL-1antibody (3.3 mg/ml), followed by gentle stirring at 4° C. for 24 hours.After the insoluble matter was removed with a filter (0.45 μm), theantibody fraction was purified by gel filtration HPLC and concentratedin the same manner as in Example 1 to give 912 μg ofNL-1-(PEG-Gly-Pro-ADM)_(m) (protein content: 0.76 mg/ml) (yield: 60%).

In the obtained conjugate, the number of molecules of adriamycin boundwas 1.5 per antibody molecule as calculated from the absorbances at 280nm and 495 nm in the same manner as in Example 1. It was confirmed thatthe affinity of the conjugate was approximately equal to that of anunbound antibody according to the fluorescent antibody method describedin Example 1.

EXAMPLE 4

Toxin Conjugate (Ia-4): KM-231-(PEG-Ala-Val-DNR)_(m)

In 500 μl of methylene chloride was dissolved 96 μg (0.08 μmol) ofCompound (XI-4) obtained in Reference Example 9, and 10 μl of a solutionof HONSu (0.38 μmol) in methylene chloride (4.3 mg/ml) and 10 μl of asolution of DCC (0.37 μmol) in methylene chloride (7.7 mg/ml) were addedsuccessively thereto under ice cooling. After stirring under ice coolingfor 1 hour and then at room temperature for 1 hour, the insoluble matter(DCU) was removed by filtration, and the solvent was removed from thefiltrate under reduced pressure. The residue was dissolved in 27 μl ofDMSO, and 114 μl of a phosphate buffer was added thereto. To theresulting mixture was added 563 μl of an aqueous solution of KM-231antibody (1.0 mg/ml), followed by gentle stirring at 4° C. for 24 hours.After the insoluble matter was removed with a filter (0.45 μm), theantibody fraction was purified by gel filtration HPLC and concentratedin the same manner as in Example 1 to give 510 μg ofKM-231-(PEG-Ala-Val-DNR)_(m) (protein content: 0.85 mg/ml) (yield: 91%).

In the obtained conjugate, the number of molecules of daunorubicin boundwas 3.1 per antibody molecule as calculated from the absorbance at 280nm and that at 495 nm (ε=1.15×10⁴ M⁻¹ cm⁻¹) in the same manner as inExample 1. It was confirmed that the affinity of the conjugate wasapproximately equal to that of an unbound antibody according to thefluorescent antibody method described in Example 1.

EXAMPLE 5

Toxin Conjugate (Ia-5): KM-231-(PEG-Ala-Pro-DNR)_(m)

In 500 μl of methylene chloride was dissolved 348 μg (0.27 μmol) ofCompound (XI-5) obtained in Reference Example 10, and 10 μl of asolution of HONSu (1.4 μmol) in methylene chloride (15.7 mg/ml) and 10μl of a solution of DCC (1.4 μmol) in methylene chloride (28.2 mg/ml)were added successively thereto under ice cooling. After stirring underice cooling for 1 hour and then at room temperature for 1 hour, theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. The residue wasdissolved in 100 μl of DMSO, and 410 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 2.05 ml of an aqueoussolution of KM-231 antibody (1.0 mg/ml), followed by gentle stirring at4° C. for 24 hours. After the insoluble matter was removed with a filter(0.45 μm), the antibody fraction was purified by gel filtration HPLC andconcentrated in the same manner as in Example 1 to give 2.0 mg ofKM-231-(PEG-Ala-Pro-DNR)_(m) (protein content: 1.36 mg/ml) (yield:100%).

In the obtained conjugate, the number of molecules of daunorubicin boundwas 1.9 per antibody molecule as calculated from the absorbances at 280nm and 495 nm in the same manner as in Example 4. It was confirmed thatthe affinity of the conjugate was approximately equal to that of anunbound antibody according to the fluorescent antibody method describedin Example 1, wherein SW1116 cells were used as the cells forevaluation.

EXAMPLE 6

Toxin Conjugate (Ia-6): KM-231-(PEG-Gly-Pro-DNR)_(m)

In 500 μl of methylene chloride was dissolved 154 μg (0.12 μmol) ofCompound (XI-6) obtained in Reference Example 11, and 10 μl of asolution of HONSu (0.61 μmol) in methylene chloride (7.0 mg/ml) and 10μl of a solution of DCC (0.61 μmol) in methylene chloride (12.6 mg/ml)were added successively thereto under ice cooling. After stirring underice cooling for 1 hour and then at room temperature for 1 hour, theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. The residue wasdissolved in 40 μl of DMSO, and 185 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 825 μl of an aqueoussolution of KM-231 antibody (1.0 mg/ml), followed by gentle stirring at4° C. for 24 hours. After the insoluble matter was removed with a filter(0.45 μm), the antibody fraction was purified by gel filtration HPLC andconcentrated in the same manner as in Example 1 to give 660 μg ofKM-231-(PEG-Gly-Pro-DNR)_(m) (protein content: 1.1 mg/ml) (yield: 80%).

In the obtained conjugate, the number of molecules of daunorubicin boundwas 1.5 per antibody molecule as calculated from the absorbances at 280nm and 495 nm in the same manner as in Example 4. It was confirmed thatthe affinity of the conjugate was approximately equal to that of anunbound antibody according to the fluorescent antibody method describedin Example 5.

EXAMPLE 7

Toxin Conjugate (Ia-7): NL-1-[PEG-Ala-Val-Compound (20)]_(m)

In 500 μl of methylene chloride was dissolved 100 μg (0.08 μmol) ofCompound (XI-7) obtained in Reference Example 12, and 10 μl of asolution of HONSu (0.41 μmol) in methylene chloride (4.7 mg/ml) and 10μl of a solution of DCC (0.41 μmol) in methylene chloride (8.4 mg/ml)were added successively thereto under ice cooling. After stirring underice cooling for 1 hour and then at room temperature for 1.5 hours, theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. The residue wasdissolved in 30 μl of DMSO, and 527 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 203 μl of an aqueoussolution of NL-1 antibody (3.0 mg/ml), followed by gentle stirring at 4°C. for 24 hours. After the insoluble matter was removed with a filter(0.45 μm), the antibody fraction was purified by gel filtration HPLC andconcentrated in the same manner as in Example 1 to give 648 μg ofNL-1-[PEG-Ala-Val-Compound (20)]_(m) (protein content: 1.1 mg/ml)(yield: 100%).

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the fluorescentantibody method described in Example 1.

The number of molecules of Compound (20) bound per antibody molecule wascalculated by subjecting the conjugate to enzyme treatment(thermolysin), and quantitatively determining released H-Val-Compound(20) by HPLC according to the method described in Reference Example 27.It was found that in the obtained conjugate, the number of molecules ofCompound (20) was 0.38 per antibody molecule.

EXAMPLE 8

Toxin Conjugate (Ia-8): NL-1-[PEG-Ala-Pro-Compound (20)]_(m)

In 500 μl of methylene chloride was dissolved 211 μg (0.17 μmol) ofCompound (XI-8) obtained in Reference Example 13, and 10 μl of asolution of HONSu (0.85 μmol) in methylene chloride (9.8 mg/ml) and 10μl of a solution of DCC (0.85 μmol) in methylene chloride (17.5 mg/ml)were added successively thereto under ice cooling. After stirring underice cooling for 1 hour and then at room temperature for 1 hour, theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. The residue wasdissolved in 60 μl of DMSO, and 1115 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 425 μl of an aqueoussolution of NL-1 antibody (3.0 mg/ml), followed by gentle stirring at 4°C. for 24 hours. After the insoluble matter was removed with a filter(0.45 μm), the antibody fraction was purified by gel filtration HPLC andconcentrated in the same manner as in Example 1 to give 1.2 mg ofNL-1-[PEG-Ala-Pro-Compound (20)]_(m) (protein content: 1.76 mg/ml)(yield: 97%).

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the fluorescentantibody method described in Example 1.

The number of molecules of Compound (20) bound per antibody molecule wascalculated by subjecting the conjugate to enzyme treatment (prolineendopeptidase), and quantitatively determining released Compound (20) byHPLC according to the method described in Reference Example 27. It wasfound that in the obtained conjugate, the number of molecules ofCompound (20) was 0.45 per antibody molecule.

EXAMPLE 9

Toxin Conjugate (Ia-9): NL-1-[PEG-Gly-Pro-Compound (20)]_(m)

In 500 μl of methylene chloride was dissolved 135 μg (0.11 μmol) ofCompound (XI-9) obtained in Reference Example 14, and 10 μl of asolution of HONSu (0.55 μmol) in methylene chloride (6.3 mg/ml) and 10μl of a solution of DCC (0.55 μmol) in methylene chloride (11.3 mg/ml)were added successively thereto under ice cooling. After stirring underice cooling for 1 hour and then at room temperature for 1 hour, theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. The residue wasdissolved in 40 μl of DMSO, and 735 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 275 μl of an aqueoussolution of NL-1 antibody (3.0 mg/ml), followed by gentle stirring at 4°C. for 24 hours. After the insoluble matter was removed with a filter(0.45 μm), the antibody fraction was purified by gel filtration HPLC andconcentrated in the same manner as in Example 1 to give 774 μg ofNL-1-[PEG-Gly-Pro-Compound (20)]_(m) (protein content: 1.2 mg/ml)(yield: 94%).

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the fluorescentantibody method described in Example 1.

The number of molecules of Compound (20) bound per antibody molecule wascalculated by subjecting the conjugate to enzyme treatment (prolineendopeptidase), and quantitatively determining released Compound (20) byHPLC according to the method described in Reference Example 27. It wasfound that in the obtained conjugate, the number of molecules ofCompound (20) was 0.49 per antibody molecule.

EXAMPLE 10

Toxin Conjugate (Ia-10): KM-231-[PEG-Ala-Val-Compound (12)]_(m)

In 0.4 ml of methanol was dissolved 0.45 mg (0.33 μmol) of Compound(X-1) obtained in Reference Example 16, and 1 mg of 10% palladium carboncatalyst was added thereto in an atmosphere of nitrogen, followed byvigorous stirring in a hydrogen stream at -15° C. for 5 hours. Afterremoval of the catalyst by filtration, the solvent was removed from thefiltrate under reduced pressure at a temperature below 0° C. to obtain0.14 mg (0.11 μmol) of HO-PEG-Ala-Val-Compound (12). The obtainedcompound (0.14 mg) was dissolved in 250 μl of a solution of HONSu inmethylene chloride (0.076 mg/ml) under ice cooling, and 250 μl of asolution of DCC in methylene chloride (0.14 mg/ml) was added thereto,followed by stirring for 2.5 hours under ice cooling. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure at a temperature below 0° C. Theresidue was dissolved in 36 μl of DMSO, and 204 μl of an ice-cooledphosphate buffer was added thereto. To the resulting mixture was added0.56 ml of an aqueous solution of KM-231 antibody (0.99 mg/ml) under icecooling, followed by gentle stirring at 4° C. for 24 hours. After theinsoluble matter was removed with a filter (0.22 μm), the antibodyfraction was purified by gel filtration HPLC and concentrated in thesame manner as in Example 1 to give 250 μg ofKM-231-[PEG-Ala-Val-Compound (12)]_(m) (protein content: 0.5 mg/ml)(yield: 5.1%).

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the fluorescentantibody method described in Example 4.

EXAMPLE 11

Toxin Conjugate (Ia-11): KM-641-[PEG-Ala-Val-Compound (12)]_(m)

In 0.3 ml of methanol was dissolved 2.5 mg (1.8 μmol) of Compound (X-1)obtained in Reference Example 16, and 1 mg of 10% palladium carboncatalyst was added thereto in an atmosphere of nitrogen, followed byvigorous stirring in a hydrogen stream at -18° C. for 4 hours. Afterremoval of the catalyst by filtration, the solvent was removed from thefiltrate under reduced pressure at a temperature below 0° C. to obtain0.05 mg (0.04 μmol) of HO-PEG-Ala-Val-Compound (12). The obtainedcompound (0.05 mg) was dissolved in 250 μl of a solution of HONSu inmethylene chloride (0.028 mg/ml) under ice cooling, and 250 μl of asolution of DCC in methylene chloride (0.05 mg/ml) was added thereto,followed by stirring for 4.5 hours under ice cooling. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure at a temperature below 0° C. Theresidue was dissolved in 15 μl of DMSO, and 80 μl of a cooled phosphatebuffer was added thereto. To the resulting mixture was added 205 μl ofan aqueous solution of KM-641 antibody (1.47 mg/ml) under ice cooling,followed by gentle stirring at 4° C. for 24 hours. After the insolublematter was removed with a filter (0.22 μm), the antibody fraction waspurified by gel filtration HPLC and concentrated in the same manner asin Example 1 to give 200 μg of KM-641-[PEG-Ala-Val-Compound (12)]_(m)(protein content: 70 μg/ml) (yield: 66%).

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the fluorescentantibody method described in Example 4.

REFERENCE EXAMPLE 1

Compound (VIII-1): BzlO-PEG-Ala-Val-OH

(a) Compound (V-1): BzlO-PEG-OH

In 100 ml of DMF was dissolved 10 g (16.7 mmol) of polyethylene glycoldicarboxylic acid [HO-PEG-OH, average molecular weight: 600 (Fluka FineChemicals Co.)], and 2.75 g of anhydrous potassium carbonate was addedthereto, followed by stirring at room temperature. To the resultingsolution was added dropwise a solution prepared by dissolving 2 ml (8.4mmol) of benzyl bromide in 100 ml of DMF, over 30 minutes, followed byfurther stirring for 24 hours. To the resulting solution was added 100ml of water, and the mixture was adjusted to pH 1-2 with iN HCl. Afterextraction was carried out six times with 100 ml portions of ethylacetate, the ethyl acetate layer was dried over anhydrous sodiumsulfate, and the solvent was removed under reduced pressure. Then, theresidue was subjected to purification using 200 ml of silica gel (WakoGel C-200), and as the developer, 200 ml each of chloroform-methanolmixtures (100:0, 50:1, 30:1, 20:1, 10:1, 5:1). The eluate was taken in10 ml fractions. The solvent was removed from the desired fractionsunder reduced pressure, the desired fractions being identified by silicagel thin layer chromatography. The residue was dissolved in a smallamount of chloroform, followed by filtration. The solvent was removedfrom the filtrate under reduced pressure to give 0.9 g (1.3 mmol) of thedesired compound, BzlO-PEG-OH (yield: 7.8%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.)Chloroform:methanol=3:1; Rf value: 0.5; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 7.36 (5H, m, C₆ H₅), 5.19 (2H, s, CH₂), 4.12 (4H, s,OCH₂), 3.64 (4nH, brs, OCH₂ CH₂)

(b) Compound (VI-1): H-Ala-Val-OtBu

In 160 ml of THF was dissolved 1.6 g (7.6 mmol) of H-Val-OtBuhydrochloride, and 1.0 ml (9.2 mmol) of NMM was added thereto, followedby stirring at room temperature. To the resulting solution was added 2.5g (7.6 mmol) of Z-Ala-ONSu, followed by further stirring at roomtemperature for 24 hours. After removal of the solvent under reducedpressure, 50 ml each of chloroform and a phosphate buffer (pH 7.0) weresuccessively added. The chloroform layer was extracted and dried oversodium sulfate, and the solvent was removed under reduced pressure toobtain an oily residue. The obtained residue was subjected topurification using 200 ml of silica gel (Wako Gel C-200), and as thedeveloper, 200 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1). The eluate was taken in 10 ml fractions. The solvent was removedfrom the desired fractions under reduced pressure, the desired fractionsbeing identified by silica gel thin layer chromatography [Kieselgel 60(Merck & Co., Inc.), chloroform:methanol=10:1, Rf value: 0.9], whereby3.0 g of Z-Ala-Val-OtBu was obtained as an oily substance. The obtainedcompound (3.0 g) was dissolved in 60 ml of THF and 60 ml of methanol,and 320 mg of 10% palladium carbon catalyst was added thereto, followedby vigorous stirring in an atmosphere of hydrogen at room temperaturefor 7 hours. The catalyst was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure to give 1.7 g (7.1mmol) of the desired compound, H-Ala-Val-OtBu (yield: 93%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.)Chloroform:methanol=10:1; Rf value: 0.4; Mass spectrum (SIMS): 245 (M+H)

(c) Compound (VIII-1): BzlO-PEG-Ala-Val-OH

In 6.0 ml of methylene chloride was dissolved 400 mg (0.58 mmol) ofBzlO-PEG-OH obtained in the above (a), and 119 mg (0.58 mmol) of DCC wasadded thereto under ice cooling, followed by stirring for 20 minutes. Tothe resulting solution was added 6.0 ml of a solution of 118 mg (0.48mmol) of H-Ala-Val-OtBu obtained in the above (b) in methylene chloride,followed by further stirring under ice cooling for 2 hours. Afterremoval of the solvent under reduced pressure, 5.0 ml of ethyl acetatewas added, followed by stirring under ice cooling for one hour. Theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. Then the residue wassubjected to purification using 50 ml of silica gel, and as thedeveloper, 100 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1). The eluate was taken in 5 ml fractions. The solvent was removedfrom the desired fractions under reduced pressure, the desired fractionsbeing identified by silica gel thin layer chromatography [Kieselgel 60(Merck & Co., Inc.), chloroform:methanol=10:1, Rf value: 0.7], whereby0.19 g of BzlO-PEG-Ala-Val-OtBu was obtained. In 4.6 ml of methylenechloride was dissolved 0.19 g of the obtained compound, and 4.6 ml ofTFA was added thereto, followed by stirring at room temperature for 24hours. After removal of the solvent under reduced pressure, the residuewas subjected to purification using 20 ml of silica gel, and as thedeveloper, 50 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 10:1, 5:1). The eluate was taken in 5 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography, whereby 0.14 g (0.16 mmol) of the desired compound,BzlO-PEG-Ala-Val-OH, was obtained (yield: 28%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.)Chloroform:methanol=10:1; Rf value: 0.2; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 7.36 (5H, m, C₆ H₅), 5.19 (2H, s, CH₂), 4.12 (4H, s,OCH₂), 3.64 (4nH, brs, OCH₂ CH₂), 3.23 [1H, s, CH(Ala)], 2.23 [1H, brq,J=6.0 Hz, CH(Val)], 1.26 [1H, s, CH(Val)], 1.17 [3H, d, J=2.8 Hz, CH₃(Ala)], 0.89 [6H, brd, J=2.5 Hz, CH₃ (Val)]

REFERENCE EXAMPLE 2

Compound (VIII-2): BzlO-PEG-Ala-Pro-OH

(a) Compound (VI-2): H-Ala-Pro-OtBu

In 65.7 ml of THF was dissolved 657 mg (3.8 mmol) of H-Pro-OtBu, and1.23 g (3.8 mmol) of Z-Ala-ONSu was added thereto, followed by stirringat room temperature for 24 hours. After removal of the solvent underreduced pressure, 50 ml each of chloroform and a phosphate buffer (pH7.0) were successively added. The chloroform layer was extracted anddried over anhydrous sodium sulfate, and the solvent was removed underreduced pressure to obtain an oily residue. The obtained residue waspurified using 200 ml of silica gel (Wako Gel C-200), and as thedeveloper, 100 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1). The solvent was removed from the desired fractions under reducedpressure, the desired fractions being identified by silica gel thinlayer chromatography [Kieselgel 60 (Merck & Co., Inc.),chloroform:methanol=10:1, Rf value: 0.8], whereby 1.47 g ofZ-Ala-Pro-OtBu was obtained as an oily substance. The obtained compound(1.47 g) was dissolved in 30 ml of THF and 30 ml of methanol, and 250 mgof 10% palladium carbon catalyst was added thereto, followed by vigorousstirring in an atmosphere of hydrogen at room temperature for 8 hours.After the catalyst was removed by filtration, the solvent was removedfrom the filtrate under reduced pressure. Then the residue was subjectedto purification using 200 ml of silica gel (Wako Gel C-200), and as thedeveloper, 100 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 10:1, 5:1). The eluate was taken in 10 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography, whereby 0.67 g (2.8 mmol) of the desired compound,H-Ala-Pro-OtBu, was obtained (yield: 74%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=10:1; Rf value: 0.2; Mass spectrum (SIMS): 243 (M+H)

(b) Compound (VIII-2): BzlO-PEG-Ala-Pro-OH

In 6.0 ml of methylene chloride was dissolved 400 mg (0.58 mmol) ofBzlO-PEG-OH obtained in Reference Example 1 (a), and 119 mg (0.58 mmol)of DCC was added thereto under ice cooling, followed by stirring for 20minutes. To the resulting solution was added 6.0 ml of a solution of 116mg (0.48 mmol) of H-Ala-Pro-OtBu obtained in the above (a) in methylenechloride, followed by further stirring under ice cooling for 2 hours.After removal of the solvent under reduced pressure, 5.0 ml of ethylacetate was added, followed by stirring under ice cooling for one hour.The insoluble matter (DCU) was removed by filtration, and the solventwas removed from the filtrate under reduced pressure to obtain 0.21 g ofa residue containing BzlO-PEG-Ala-Pro-OtBu. The obtained residue wasdissolved in 5.1 ml of methylene chloride, and 5.1 ml of TFA was addedthereto, followed by stirring at room temperature for 24 hours. Afterremoval of the solvent under reduced pressure, the residue was subjectedto purification using 20 ml of silica gel (Wako Gel C-200), and as thedeveloper, 50 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 10:1, 5:1, 3:1). The eluate was taken in 5 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography, whereby 164 mg (0.19 mmol) of the desired compound,BzlO-PEG-Ala-Pro-OH, was obtained (yield: 33%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=5:1; Rf value: 0.2; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 7.36 (5H, m, C₆ H₅), 5.19 (2H, s, CH₂), 4.40 [2H, br,CH₂ (Pro)], 4.12 (4H, s, OCH₂), 3.80 [1H, q, J=6.0 Hz, CH(Ala)], 3.64(4nH, brs, OCH₂ CH₂), 3.59 [2H, br, CH₂ (Pro)], 2.36 [1H, br, CH(Pro)],2.02 [2H, br, CH₂ (Pro)], 1.29 [3H, brd, J=3.5 Hz, CH₃ (Ala)]

REFERENCE EXAMPLE 3

Compound (VIII-3): BzlO-PEG-Gly-Pro-OH

(a) Compound (VI-3): H-Gly-Pro-OtBu

In 100 ml of THF was dissolved 1.0 g (5.8 mmol) of H-Pro-OtBu, and 1.8 g(5.8 mmol) of Z-Gly-ONSu was added thereto, followed by stirring at roomtemperature for 24 hours. After removal of the solvent under reducedpressure, 50 ml each of chloroform and a phosphate buffer (pH 7.0) weresuccessively added. The chloroform layer was extracted and dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure to obtain an oily residue. The obtained residue was subjectedto purification using 200 ml of silica gel (Wako Gel C-200), and as thedeveloper, 150 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1). The eluate was taken in 10 ml fractions. The solvent was removedfrom the desired fractions under reduced pressure, the desired fractionsbeing identified by silica gel thin layer chromatography [Kieselgel 60(Merck & Co., Inc.), chloroform:methanol=10:1, Rf value: 0.9], whereby1.93 g of Z-Gly-Pro-OtBu was obtained as an oily substance. The obtainedcompound (1.93 g) was dissolved in 20 ml of THF and 40 ml of methanol,and 440 mg of 10% palladium carbon catalyst was added thereto, followedby vigorous stirring in an atmosphere of hydrogen at room temperaturefor 15 hours. After the catalyst was removed by filtration, the solventwas removed from the filtrate under reduced pressure to give 1.13 g (4.9mmol) of the desired compound, H-Gly-Pro-OtBu (yield: 86%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=10:1; Rf value: 0.2; Mass spectrum (SIMS): 211 (M+H)

(b) Compound (VIII-3): BzlO-PEG-Gly-Pro-OH

In 6.0 ml of methylene chloride was dissolved 400 mg (0.58 mmol) ofBzlO-PEG-OH obtained in Reference Example 1 (a), and 119 mg of DCC wasadded thereto under ice cooling, followed by stirring for 20 minutes. Tothe resulting solution was added 6.0 ml of a solution of 110 mg (0.48mmol) of H-Gly-Pro-OtBu obtained in the above (a) in methylene chloride,followed by further stirring under ice cooling for 2 hours. Afterremoval of the solvent under reduced pressure, 5.0 ml of ethyl acetatewas added, followed by stirring under ice cooling for one hour. Theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. Then the residue wassubjected to purification using 50 ml of silica gel (Wako Gel C-200),and as the developer, 100 ml each of chloroform-methanol mixtures(100:0, 100:1, 50:1). The eluate was taken in 10 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography [Kieselgel 60 (Merck & Co., Inc.),chloroform:methanol=10:1, Rf value: 0.7], whereby 0.21 g ofBzlO-PEG-Gly-Pro-OtBu was obtained. In 5.1 ml of methylene chloride wasdissolved 0.21 g of the obtained compound, and 5.1 ml of TFA was addedthereto, followed by stirring at room temperature for 24 hours. Afterremoval of the solvent under reduced pressure, the residue was subjectedto purification using 20 ml of silica gel (Wako Gel C-200), and as thedeveloper, 50 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 20:1, 10:1, 5:1, 3:1). The eluate was taken in 5 mlfractions. The solvent was removed from the desired fractions underreduced pressure, the desired fractions being identified by silica gelthin layer chromatography, whereby 164 mg (0.2 mmol) of the desiredcompound, BzlO-PEG-Gly-Pro-OH, was obtained (yield: 33%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=10:1; Rf value: 0.2; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 7.36 (5H, m, C₆ H₅), 5.19 (2H, s, CH₂), 4.40 [2H, br,CH₂ (Pro)], 4.12 (4H, s, OCH₂), 3.82 [2H, s, CH₂ (Gly)], 3.64 (4nH, brs,OCH₂ CH₂), 3.59 [2H, br, CH₂ (Pro)], 2.21 [1H, s, CH(Pro)], 2.02 [2H,br, CH₂ (Pro)]

REFERENCE EXAMPLE 4

Compound (VIII-4): PicO-PEG-Gly-Pro-OH

(a) Compound (V-2): PicO-PEG-OH

In 50 ml of DMF was dissolved 10 g (16.7 mmol) of HO-PEG-OH, and 1.37 g(8.4 mmol) of picolyl chloride hydrochloride and 3.4 ml (25.1 mmol) oftriethylamine were successively added thereto, followed by stirring at90 to 100° C. for 2 hours. Then, the solvent was removed under reducedpressure to obtain a mixture of polyethylene glycol dicarboxylic acid(unreacted), monopicolyl ester and dipicolyl ester. In 100 ml ofchloroform was dissolved 1.97 g of this reaction mixture, and 100 ml ofwater was added thereto, followed by addition of 1N sodium hydroxide toadjust the water layer to pH 9.5. After the water layer was extractedand adjusted to pH 6.5 with 1N HCl, extraction was carried out 6 to 7times with 100 ml portions of chloroform. During the repeatedextractions, the pH of the water layer was kept at 6.5 with 1N HCl.After the chloroform layer was dried over anhydrous sodium sulfate, thesolvent was removed under reduced pressure to give 1.0 g (1.5 mmol) ofthe desired compound, PicO-PEG-OH (yield: 8.9%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=3:1; Rf value: 0.5; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 5.21 (2H, s, CH₂), 4.12 (4H, s, OCH₂), 3.64 (4nH, brs,OCH₂ CH₂), 7.28 [2H, d, J=3.5 Hz, H-3, H-5(Pic)], 8.65 [2H, d, J=3.5 Hz,H-2, H-6(Pic)]

(b) Compound (VIII-4): PicO-PEG-Gly-Pro-OH

In 8 ml of methylene chloride was dissolved 500 mg (0.73 mmol) ofPicO-PEG-OH obtained in the above (a), and 151 mg (0.73 mmol) of DCC wasadded thereto under ice cooling, followed by stirring for 20 minutes. Tothe resulting solution was added 8 ml of a solution of 139 mg (0.61mmol) of H-Gly-Pro-OtBu obtained in Reference Example 3 (a) in methylenechloride, followed by stirring under ice cooling for 3 hours. Afterremoval of the solvent under reduced pressure, 5 ml of ethyl acetate wasadded, followed by stirring under ice cooling for one hour. Theinsoluble matter (DCU) was removed by filtration, and the solvent wasremoved from the filtrate under reduced pressure. Then the residue wassubjected to purification using 50 ml of silica gel (Wako Gel C-200),and as the developer, 50 ml each of chloroform-methanol mixtures (100:0,100:1, 50:1, 30:1, 20:1, 10:1). The eluate was taken in 5 ml fractions.The solvent was removed from the desired fractions under reducedpressure, the desired fractions being identified by silica gel thinlayer chromatography [Kieselgel 60 (Merck & Co., Inc.),chloroform:methanol=10:1, Rf value: 0.5], whereby 374 mg ofPicO-PEG-Gly-Pro-OtBu was obtained. In 9.0 ml of methylene chloride wasdissolved 374 mg of the obtained compound, and 9.0 ml of TFA was addedthereto, followed by stirring at room temperature for 24 hours. Afterremoval of the solvent under reduced pressure, the residue was subjectedto purification using 20 ml of silica gel (Wako Gel C-200), and as thedeveloper, 50 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 10:1, 5:1). The eluate was taken in 5 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography, whereby 131 mg (0.16 mmol) of the desired compound,PicO-PEG-Gly-Pro-OH, was obtained (yield: 26%)

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=5:1; Rf value: 0.1; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 5.21 (2H, s, CH₂), 4.40 [2H, br, CH₂ (Pro)], 4.12 (4H,s, OCH₂), 3.82 [2H, s, CH₂ (Gly)], 3.64 (4nH, brs, OCH₂ CH₂), 3.59 [2H,br, CH₂ (Pro)], 2.21 [1H, s, CH(Pro)], 2.02 [2H, br, CH₂ (Pro)], 7.28[2H, d, J=3.5 Hz, H-3, H-5(Pic)], 8.65 [2H, d, J=3.5 Hz, H-2, H-6(Pic)]

REFERENCE EXAMPLE 5

Compound (VIII-5): PicO-PEG-Ala-Val-OH

In 7 ml of methylene chloride was dissolved 459 mg (0.67 mmol) ofPicO-PEG-OH obtained in Reference Example 4 (a), and 138 mg (0.80 mmol)of DCC was added thereto under ice cooling, followed by stirring for 20minutes. To the resulting solution was added 7 ml of a solution of 134mg (0.55 mmol) of H-Ala-Val-OtBu obtained in Reference Example 1 (b) inmethylene chloride, followed by further stirring under ice cooling for 3hours. After removal of the solvent under reduced pressure, 5 ml ofethyl acetate was added, followed by stirring under ice cooling for onehour. The insoluble matter (DCU) was removed by filtration, and thesolvent was removed from the filtrate under reduced pressure. Then theresidue was subjected to purification using 50 ml of silica gel (WakoGel C-200), and as the developer, 50 ml each of chloroform-methanolmixtures (100:0, 100:1, 50:1, 30:1, 20:1). The eluate was taken in 5 mlfractions. The solvent was removed from the desired fractions underreduced pressure, the desired fractions being identified by silica gelthin layer chromatography [Kieselgel 60 (Merck & Co., Inc.),chloroform:methanol=10:1, Rf value: 0.4], whereby 328 mg ofPicO-PEG-Ala-Val-OtBu was obtained. In 8.0 ml of methylene chloride wasdissolved 328 mg of the obtained compound, and 8.0 ml of TFA was addedthereto, followed by stirring at room temperature for 24 hours. Afterremoval of the solvent under reduced pressure, the residue was subjectedto purification using 20 ml of silica gel (Wako Gel C-200), and as thedeveloper, 50 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 10:1, 5:1). The eluate was taken in 5 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure,the desired fractions being identified by silica gel thin layerchromatography, whereby 131 mg (0.15 mmol) of the desired compound,PicO-PEG-Gly-Pro-OH, was obtained (yield: 27%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=5:1; Rf value: 0.1; ¹ HNMR spectrum (100 MHz, inCDCl₃) δ (ppm): 5.21 (2H, s, CH₂), 4.12 (4H, s, OCH₂), 3.64 (4nH, brs,OCH₂ CH₂), 3.23 [1H, s, CH(Ala)], 2.23 [1H, brq, J=6.0 Hz, CH(Val)],7.28 [2H, d, J=3.5 Hz, H-3, H-5(Pic)], 1.26 [1H, s, CH(Val)], 1.17 [3H,d, J=2.8 Hz, CH₃ (Ala)], 8.65 [2H, d, J=3.5 Hz, H-2, H-6(Pic)], 0.89[6H, q, J=2.5 Hz, CH₃ (Val)]

REFERENCE EXAMPLE 6

Compound (XI-1): HO-PEG-Ala-Val-ADM

In 0.5 ml of methylene chloride was dissolved 11.4 mg (13.3 μmol) ofCompound (VIII-1) obtained in Reference Example 1, and 3.4 mg (29.5μmol) of HONSu and 6.2 mg (30.1 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling a solutionprepared by dissolving 0.25 mg (0.46 μmol) of adriamycin hydrochloridein 383 μl of a solution of triethylamine in dry DMF (8.5 μg/ml). Afterthe resulting mixture was allowed to stand under ice cooling for 30minutes, the solvent was removed under reduced pressure. The residue wassubjected to purification using 3.0 ml of silica gel (Wako Gel C-200).The by-product, BzlO-PEG-Ala-Val-ADM, was removed by elution using, asthe developer, 10 ml each of chloroform-methanol mixtures (10:1, 7:1,5:1, 3:1, 2:1). Then, the desired HO-PEG-Ala-Val-ADM was eluted with 20ml of a mixture of chloroform:methanol:water (13:6:1). The solvent wasremoved from the desired fraction under reduced pressure to give 179 μg(0.20 μmol) of HO-PEG-Ala-Val-ADM (yield from adriamycin: 44%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Val moiety 4.19 (4H, s, OCH₂), 3.48(4nH, brs, OCH₂ CH₂), 3.22 [1H, brs, CH(Ala)], 2.23 [1H, brq, J=6.0 Hz,CH(Val)], 1.25 [1H, s, CH(Val)], 1.17 [3H, d, J=2.8 Hz, CH₃ (Ala)], 0.89[6H, brm, CH₃ (Val)], Adriamycin moiety 7.99 (2H, d, J=6.0 Hz, H-1,H-2), 7.73 (1H, brm, H-3), 5.43 (1H, brm, H-1'), 5.42 (1H, d, J=3.7 Hz,H-14b), 5.35 (1H, d, J=3.7 Hz, H-1'), 5.14 (1H, brs, H-7), 4.34 (1H, q,J=6.5 Hz, H-5'), 4.10 (3H, s, 4-OCH₃), 3.22 (1H, d, J=18 Hz, H-10b),3.10 (1H, d, J=18 Hz, H-10a), 2.50 (1H, brd, J=14 Hz, H-8b), 2.23 (1H,d, J=14 Hz, H-8a), 2.06 (1H, brm, H-2'b), 1.89 (1H, brm, H-2'a), 1.28(3H, d, J=6.5 Hz, 5'-CH₃); Ultraviolet absorption spectrum (in methanol,λ_(max)): 232, 274, 495, 534, 575 nm; Infrared absorption spectrum (inchloroform): 3580, 3400, 3000, 2924, 2870, 1786, 1717, 1660, 1605, 1520,1450, 1295, 1105 cm⁻¹

REFERENCE EXAMPLE 7

Compound (XI-2): HO-PEG-Ala-Pro-ADM

In 0.5 ml of methylene chloride was dissolved 9.5 mg (11.1 μmol) ofCompound (VIII-2) obtained in Reference Example 2, and 3.4 mg (29.5μmol) of HONSu and 6.2 mg (30.1 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling a solutionprepared by dissolving 0.25 mg (0.46 μmol) of adriamycin hydrochloridein 383 μl of a solution of triethylamine in dry DMF (8.5 μg/ml). Afterthe resulting mixture was allowed to stand under ice cooling for 30minutes, the solvent was removed under reduced pressure. The residue wassubjected to purification using 3.0 ml of silica gel (Wako Gel C-200).The by-product, BzlO-PEG-Ala-Pro-ADM, was removed by elution using, asthe developer, 10 ml each of chloroform-methanol mixtures (10:1, 7:1,5:1, 3:1, 2:1). Then, the desired HO-PEG-Ala-Pro-ADM was eluted with 20ml of a mixture of chloroform:methanol:water (13:6:1). The solvent wasremoved from the desired fraction under reduced pressure to give 182 μg(0.21 μmol) of HO-PEG-Ala-Pro-ADM (yield from adriamycin: 46%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Pro moiety 4.52 [2H, brm, CH₂ (Pro)],4.12 (4H, s, OCH₂), 3.75 [1H, q, J=6.0 Hz, CH(Ala)], 3.64 (4nH, brs,OCH₂ CH₂), 3.55 [2H, br, CH₂ (Pro)], 2.34 [1H, br, CH(Pro)], 2.04 [2H,brm, CH₂ (Pro)], 1.15 [3H, br, CH₃ (Ala)], Adriamycin moiety 8.06 (2H,d, J=6.0 Hz, H-1, H-2), 7.80 (1H, brm, H-3), 5.54 (1H, brm, H-1'), 5.51(1H, d, J=3.5 Hz, H-14b), 5.29 (1H, d, J=3.5 Hz, H-14a), 5.19 (1H, brs,H-7), 4.31 (1H, t, J=6.5 Hz, H-5'), 4.13 (3H, s, 4-OCH₃), 3.41 (1H, d,J=18 Hz, H-10b), 3.07 (1H, d, J=18 Hz, H-10a), 2.59 (1H, brd, J=14 Hz,H-8b), 2.27 (1H, d, J=14 Hz, H-8a), 2.14 (1H, br, H-2'b), 1.82 (1H, br,H-2'a), 1.26 (3H, d, J=6.5 Hz, 5'--CH₃); Ultraviolet absorption spectrum(in methanol, λ_(max)): 233, 252, 290, 470, 495, 534, 578 nm; Infraredabsorption spectrum (in chloroform): 3580, 3400, 3005, 2930, 2870, 1780,1718, 1658, 1580, 1450, 1282, 1110 cm⁻¹

REFERENCE EXAMPLE 8

Compound (XI-3): HO-PEG-Gly-Pro-ADM

In 0.5 ml of methylene chloride was dissolved 10.8 mg (12.5 μmol) ofCompound (VIII-3) obtained in Reference Example 3, and 3.4 mg (29.5μmol) of HONSu and 6.2 mg (30.1 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling a solutionprepared by dissolving 0.175 mg (0.3 μmol) of adriamycin hydrochloridein 250 μl of a solution of triethylamine in dry DMF (8.5 μg/ml). Afterthe resulting mixture was allowed to stand under ice cooling for 30minutes, the solvent was removed under reduced pressure. The residue wassubjected to purification using 5.0 ml of silica gel (Wako Gel C-200).The by-product, BzlO-PEG-Gly-Pro-ADM, was removed by elution using, asthe developer, 10 ml each of chloroform-methanol mixtures (5:1, 3:1,2:1). Then, the desired HO-PEG-Gly-Pro-ADM was eluted with 20 ml of amixture of chloroform:methanol:water (13:6:1). The solvent was removedfrom the desired fraction under reduced pressure to give 240 μg (0.19μmol) of HO-PEG-Gly-Pro-ADM (yield from adriamycin: 62%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Gly-Pro moiety 4.22 [2H, brm, CH₂ (Pro)],4.17 (4H, m, OCH₂), 4.08 [2H, s, CH₂ (Gly)], 3.64 (4nH, brs, OCH₂ CH₂),3.52 [2H, br, CH₂ (Pro)], 2.22 [1H, br, CH(Pro)], 1.95 [2H, brm, CH₂(Pro)], Adriamycin moiety 8.05 (2H, m, H-1, H-2), 7.77 (1H, m, H-3),5.50 (1H, d, J=3.7 Hz, H-1'), 5.34 (1H, d, J=3.5 Hz, H-14b), 5.30 (1H,d, J=3.8 Hz, H-1'), 5.19 (1H, brs, H-7), 4.31 (1H, q, J=6.8 Hz, H-5'),4.10 (3H, s, 4-OCH₃), 3.30 (1H, d, J=18 Hz, H-10b), 3.03 (1H, d, J=18Hz, H-10a), 2.48 (1H, brd, J=14 Hz, H-8b), 2.20 (1H, d, J=14 Hz, H-8a),2.04 (1H, brm, H-2'b), 1.83 (1H, m, H-2'a), 1.14 (3H, brd, J=7 Hz,5'--CH₃); Ultraviolet absorption spectrum (in methanol, λ_(max)): 233,250, 290, 470, 495, 530, 576 nm; Infrared absorption spectrum (inchloroform): 3590, 3400, 3000, 2940, 2860, 1720, 1650, 1450, 1115 cm⁻¹

REFERENCE EXAMPLE 9

Compound (XI-4): HO-PEG-Ala-Val-DNR

In 0.5 ml of methylene chloride was dissolved 11.4 mg (13.3 μmol) ofCompound (VIII-1) obtained in Reference Example 1, and 3.6 mg (31.3μmol) of HONSu and 6.4 mg (31.0 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling a solutionprepared by dissolving 0.90 mg (1.7 μmol) of daunorubicin hydrochloridein 500 μl of a solution of triethylamine in dry DMF (0.48 μg/ml). Afterthe resulting mixture was allowed to stand under ice cooling for 30minutes and then at room temperature for one hour, the solvent wasremoved under reduced pressure. The residue was subjected topurification using 3.0 ml of silica gel (Wako Gel C-200). Theby-product, BzlO-PEG-Ala-Val-DNR, was removed by elution using, as thedeveloper, 10 ml each of chloroform-methanol mixtures (10:1, 7:1, 5:1,3:1, 2:1). Then, the desired HO-PEG-Ala-Val-DNR was eluted with 20 ml ofa mixture of chloroform:methanol:water (13:6:1). The solvent was removedfrom the desired fraction under reduced pressure to give 38 μg (0.03μmol) of HO-PEG-Ala-Val-DNR (yield from daunorubicin: 1.8%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Val moiety 4.19 (4H, s, OCH₂), 3.49(4nH, brs, OCH₂ CH₂), 3.20 [1H, brs, CH(Ala)], 2.29 [1H, brq, J=5.7 Hz,CH(Val)], 1.29 [1H, s, CH(Val)], 1.24 [3H, d, J=2.8 Hz, CH₃ (Ala)], 0.95[6H, brm, CH₃ (Val)], Daunorubicin moiety 7.82 (2H, m, H-1, H-2), 7.72(1H, m, H-3), 5.34 (1H, d, J=3.7 Hz, H-1'), 5.27 (1H, brs, H-7), 4.31(1H, q, J=6.5 Hz, H-5'), 4.19 (3H, s, 4-OCH₃), 3.29 (1H, d, J=18 Hz,H-10b), 3.12 (1H, d, J=18 Hz, H-10a), 2.80 (3H, s, H-14), 2.34 (1H, brd,J=14 Hz, H-8b), 2.22 (1H, d, J=14 Hz, H-8a), 1.91 (1H, brm, H-2'b), 1.83(1H, brm, H-2'a), 1.29 (3H, d, J=6.5 Hz, 5'-CH₃); Ultraviolet absorptionspectrum (in methanol, λ_(max)): 232, 274, 495, 534, 575 nm; Infraredabsorption spectrum (in chloroform): 3580, 3400, 3000, 2924, 2870, 1786,1717, 1660, 1605, 1520, 1450, 1295, 1105 cm⁻¹

REFERENCE EXAMPLE 10

Compound (XI-5): HO-PEG-Ala-Pro-DNR

In 0.5 ml of methylene chloride was dissolved 9.7 mg (11.3 μmol) ofCompound (VIII-2) obtained in Reference Example 2, and 3.1 mg (26.9μmol) of HONSu and 5.5 mg (26.7 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling 165 μl (0.5μmol) of a solution prepared by dissolving 0.9 mg of daunorubicinhydrochloride in 500 μl of a solution of triethylamine in dry DMF (0.48μg/ml). After the resulting mixture was allowed to stand under icecooling for 30 minutes and then at room temperature for one hour, thesolvent was removed under reduced pressure. The residue was subjected topurification using 5.0 ml of silica gel (Wako Gel C-200). Theby-product, BzlO-PEG-Ala-Pro-DNR, was removed by elution using, as thedeveloper, 10 ml each of chloroform-methanol mixtures (10:1, 7:1, 5:1,3:1, 2:1). Then, the desired HO-PEG-Ala-Pro-DNR was eluted with 20 ml ofa mixture of chloroform:methanol:water (13:6:1). The solvent was removedfrom the desired fraction under reduced pressure to give 150 μg (0.1μmol) of HO-PEG-Ala-Pro-DNR (yield from daunorubicin: 20%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Pro moiety 4.52 [2H, br, CH₂ (Pro)],4.12 (4H, s, OCH₂), 3.75 [1H, q, J=6.0 Hz, CH(Ala)], 3.64 (4nH, brs,OCH₂ CH₂), 3.42 [2H, br, CH₂ (Pro)], 2.37 [1H, br, CH(Pro)], 2.08 [2H,brm, CH₂ (Pro)], 1.29 [3H, br, CH₃ (Ala)], Daunorubicin moiety 8.03 (2H,m, H-1, H-2), 7.72 (1H, m, H-3), 5.52 (1H, brd, J=3.7 Hz, H-1'), 5.36(1H, brm, H-1'), 5.29 (1H, brs, H-7), 4.34 (1H, q, J=6.5 Hz, H-5'), 4.09(3H, s, 4-OCH₃), 3.23 (1H, d, J=18 Hz, H-10b), 3.01 (1H, d, J=18 Hz,H-10a), 2.80 (3H, s, H-14), 2.42 (1H, brd, J=14 Hz, H-8b), 2.23 (1H, d,J=14 Hz, H-8a), 2.06 (1H, brm, H-2'b), 1.87 (1H, brm, H-2'a), 1.29 (3H,d, J=6.5 Hz, 5'-CH₃); Ultraviolet absorption spectrum (in methanol,λ_(max)): 232, 251, 290, 469, 495, 532, 580 nm; Infrared absorptionspectrum (in chloroform): 3690, 3580, 3400, 3015, 2936, 2878, 1782,1714, 1650, 1600, 1525, 1457, 1432, 1350, 1285, 1240, 1108 cm⁻¹

REFERENCE EXAMPLE 11

Compound (XI-6): HO-PEG-Gly-Pro-DNR

In 0.5 ml of methylene chloride was dissolved 11.0 mg (12.8 μmol) ofCompound (VIII-3) obtained in Reference Example 3, and 3.5 mg (30.4μmol) of HONSu and 6.3 mg (30.5 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. To the residue was added under ice cooling 156 μl (0.5μmol) of a solution prepared by dissolving 0.9 mg of daunorubicinhydrochloride in 500 μl of a solution of triethylamine in dry DMF (0.48μg/ml). After the resulting mixture was allowed to stand under icecooling for 30 minutes and then at room temperature for one hour, thesolvent was removed under reduced pressure. The residue was subjected topurification using 5.0 ml of silica gel (Wako Gel C-200). Theby-product, BzlO-PEG-Gly-Pro-DNR, was removed by elution using, as thedeveloper, 10 ml each of chloroform-methanol mixtures (10:1, 7:1, 5:1,3:1, 2:1). Then, the desired HO-PEG-Gly-Pro-DNR was eluted with 20 ml ofa mixture of chloroform:methanol:water (13:6:1). The solvent was removedfrom the desired fraction under reduced pressure to give 180 μg (0.1μmol) of HO-PEG-Gly-Pro-DNR (yield from daunorubicin: 26%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Gly-Pro moiety 4.51 [2H, brm, CH₂ (Pro)],4.17 (4H, m, OCH₂ O), 3.64 (4nH, brs, OCH₂ CH₂), 3.56 [2H, s, CH₂(Gly)], 3.50 [2H, m, CH₂ (Pro)], 2.21 [1H, s, CH(Pro)], 2.02 [2H, brm,CH₂ (Pro)], Daunorubicin moiety 8.04 (2H, m, H-1, H-2), 7.79 (1H, m,H-3), 5.34 (1H, d, J=3.7 Hz, H-1'), 5.27 (1H, brs, H-7), 4.52 (1H, q,J=6.7 Hz, H-5'), 4.12 (3H, s, 4-OCH₃), 3.22 (1H, d, J=18 Hz, H-10b),2.99 (1H, d, J=18 Hz, H-10a), 2.90 (3H, s, H-14), 2.35 (1H, brd, J=14Hz, H-8b), 2.22 (1H, d, J=14 Hz, H-8a), 2.02 (1H, brm, H-2'b), 1.18 (3H,d, J=6.6 Hz, 5'-CH₃); Ultraviolet absorption spectrum (in methanol,λ_(max)): 235, 252, 289, 470, 495, 534, 578 nm; Infrared absorptionspectrum (in chloroform): 3580, 3000, 2930, 2880, 1790, 1719, 1658,1610, 1450, 1404, 1350, 1290, 1110 cm⁻¹

REFERENCE EXAMPLE 12

Compound (XI-7): HO-PEG-Ala-Val-Compound (20)

In 0.5 ml of methylene chloride was dissolved 11.5 mg (13.4 μmol) ofCompound (VIII-1) obtained in Reference Example 1, and 3.6 mg (31.3μmol) of HONSu and 6.4 mg (31.0 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 2.5 hours. After removal ofthe catalyst by filtration, the solvent was removed from the filtrateunder reduced pressure. The residue was dissolved in a small amount ofchloroform and subjected to purification using 3.0 ml of silica gel(Wako Gel C-200). The benzyl ester form, BzlO-PEG-Ala-Val-ONSu, wasremoved by elution using, as the developer, 10 ml each ofchloroform-methanol mixtures (20:1, 10:1). Then, a solution prepared bydissolving 0.05 mg (0.10 μmol) of Compound (20) obtained in ReferenceExample 17 in 200 μl of dry DMF was charged into the column. After 150μl of dry DMF was further applied to the column, the mixture wassubjected to reaction at room temperature for 30 minutes. After washingof the column using 10 ml each of chloroform-methanol mixtures (10:1,5:1, 2:1) as the developer, the desired HO-PEG-Ala-Val-Compound (20) waseluted with 10 ml of a mixture of chloroform:methanol:water (13:6:1).The solvent was removed from the desired fraction under reduced pressureto give 112 μg (0.09 μmol) of HO-PEG-Ala-Val-Compound (20) [yield fromCompound (20): 88%].

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Val moiety 4.12 (4H, s, OCH₂), 3.64(4nH, brs, OCH₂ CH₂), 3.23 [1H, s, CH(Ala)], 2.23 (1H, brq, J=6.0 Hz,CH(Val)], 1.26 [1H, s, CH(Val)], 1.17 [3H, d, J=2.8 Hz, CH₃ (Ala)], 0.89[6H, q, J=2.5 Hz, CH₃ (Val)], Compound (20) moiety 10.57 (1H, brs), 7.68(1H, d, J=15.5 Hz), 7.12 (1H, dd, J=8.3, 2.0 Hz), 6.99 (1H, d, J=2.0Hz), 6.80 (1H, d, J=8.1 Hz), 4.40 (1H, m), 4.27 (2H, m), 4.16 (1H, brd,J=11.2 Hz), 3.90 (3H, s), 3.82 (3H, s), 3.67 (1H, m), 3.12 (2H, t, J=7.1Hz), 2.63 (3H, s), 2.38 (1H, dd, J=7.5, 3.4 Hz), 1.52 (2H, m), 1.37 (1H,t, J=4.2 Hz); Infrared absorption spectrum (in chloroform): 3690, 3592,3400, 3000, 2900, 1712, 1650, 1600, 1450, 1300, 1110 cm⁻¹

REFERENCE EXAMPLE 13

Compound (XI-8): HO-PEG-Ala-Pro-Compound (20)

In 0.5 ml of methylene chloride was dissolved 9.8 mg (11.4 μmol) ofCompound (VIII-2) obtained in Reference Example 2, and 3.2 mg (27.8μmol) of HONSu and 5.6 mg (27.1 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 3 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. The residue was dissolved in a small amount ofchloroform and subjected to purification using 3.0 ml of silica gel(Wako Gel C-200). The benzyl ester form, BzlO-PEG-Ala-Pro-ONSu, wasremoved by elution using, as the developer, 10 ml each ofchloroform-methanol mixtures (20:1, 10:1). Then, a solution prepared bydissolving 0.05 mg (0.10 μmol) of Compound (20) obtained in ReferenceExample 17 in 200 μl of dry DMF was charged into the column. After 100μl of dry DMF was further applied to the column, the mixture wassubjected to reaction at room temperature for 30 minutes. After washingof the column using 10 ml each of chloroform-methanol mixtures (10:1,2:1) as the developer, the desired HO-PEG-Ala-Pro-Compound (20) waseluted with 10 ml of a mixture of chloroform:methanol:water (13:6:1).The solvent was removed from the desired fraction under reduced pressureto give 233 μg (0.10 μmol) of HO-PEG-Ala-Pro-Compound (20) [yield fromCompound (20): 100%].

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Ala-Pro moiety 4.52 [2H, brm, CH₂ (Pro)],4.12 (4H, s, OCH₂), 3.64 (4nH, brs, OCH₂ CH₂), 3.42 (2H, br, CH₂ (Pro)],3.23 [1H, s, CH(Ala)], 2.37 [1H, br, CH(Pro)], 2.08 [2H, brm, CH₂(Pro)], 1.17 [3H, d, J=2.8 Hz, CH₃ (Ala)], Compound (20) moiety 10.23(1H, brs), 7.68 (1H, d, J=15.5 Hz), 7.10 (1H, dd, J=8.3, 2.0 Hz), 6.89(1H, d, J=2.0 Hz), 6.80 (1H, d, J=8.1 Hz), 4.40 (1H, m), 4.29 (2H, m),4.20 (1H, brd, J=11.2 Hz), 3.89 (3H, s), 3.82 (3H, s), 3.67 (1H, m),3.12 (2H, t, J=7.1 Hz), 2.63 (3H, s), 2.33 (1H, dd, J=7.5, 3.4 Hz), 1.52(2H, m), 1.35 (1H, t, J=4.2 Hz); Infrared absorption spectrum (inchloroform): 3690, 3592, 3400, 3000, 2900, 1712, 1650, 1600, 1450, 1300,1110 cm⁻¹

REFERENCE EXAMPLE 14

Compound (XI-9): HO-PEG-Gly-Pro-Compound (20)

In 0.5 ml of methylene chloride was dissolved 11.2 mg (13.0 μml) ofCompound (VIII-3) obtained in Reference Example 3, and 3.6 mg (31.3μmol) of HONSu and 6.4 mg (31.0 μmol) of DCC were added thereto underice cooling, followed by stirring at 0° C. for 2 hours. The insolublematter (DCU) was removed by filtration, and the solvent was removed fromthe filtrate under reduced pressure. Then the residue was dissolved in300 μl of methanol in a stream of nitrogen, and 2-3 mg of 10% palladiumcarbon catalyst was added thereto, followed by vigorous stirring in astream of hydrogen at room temperature for 3 hours. After removal of thecatalyst by filtration, the solvent was removed from the filtrate underreduced pressure. The residue was dissolved in a small amount ofchloroform and subjected to purification using 3.0 ml of silica gel(Wako Gel C-200). The benzyl ester form, BzlO-PEG-Gly-Pro-ONSu, wasremoved by elution using, as the developer, 10 ml each ofchloroform-methanol mixtures (20:1, 10:1). Then, a solution prepared bydissolving 0.05 mg (0.10 μmol) of Compound (20) obtained in ReferenceExample 17 in 200 μl of dry DMF was charged into the column. After 150μl of dry DMF was further applied to the column, the mixture wassubjected to reaction at room temperature for 30 minutes. After washingof the column using 10 ml each of chloroform-methanol mixtures (10:1,2:1) as the developer, the desired HO-PEG-Gly-Pro-Compound (20) waseluted with 10 ml of a mixture of chloroform:methanol:water (13:6:1).The solvent was removed from the desired fraction under reduced pressureto give 135 μg (0.10 μmol) of HO-PEG-Gly-Pro-Compound (20) [yield fromCompound (20): 100%].

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol:water=13:6:1; Rf value: 0.6; ¹ HNMR spectrum (500MHz, in CDCl₃) δ (ppm): PEG-Gly-Pro moiety 4.22 [2H, brm, CH₂ (Pro)],4.17 (4H, m, OCH₂), 4.08 [2H, s, CH₂ (Gly)], 3.64 (4nH, brs, OCH₂ CH₂),3.52 [2H, br, CH₂ (Pro)], 2.22 [1H, br, CH(Pro)], 1.95 [2H, brm, CH₂(Pro)], Compound (20) moiety 9.97 (1H, brs), 7.54 (1H, d, J=15.5 Hz),7.22 (1H, dd, J=8.3, 2.0 Hz), 6.99 (1H, d, J=2.0 Hz), 6.88 (1H, d, J=8.1Hz), 4.40 (1H, m), 4.27 (2H, m), 4.16 (1H, brd, J=11.2 Hz), 3.90 (3H,s), 3.82 (3H, s), 3.70 (1H, m), 3.12 (2H, t, J=7.1 Hz), 2.63 (3H, s),2.54 (1H, dd, J=7.5, 3.4 Hz), 1.62 (2H, m), 1.35 (1H, t, J=4.2 Hz);Infrared absorption spectrum (in chloroform): 3690, 3592, 3400, 3000,2900, 1712, 1650, 1600, 1450, 1300, 1110 cm⁻¹

REFERENCE EXAMPLE 15

Compound (12)

Compound (12) was synthesized according to the following reaction steps.##STR22##

In 30 ml of DMF was dissolved 3.0 g (16 mmol) of4-hydroxy-3,5-dimethoxybenzaldehyde [Compound (1)], and 3.4 g ofanhydrous potassium carbonate was added thereto. To the resultingsolution was added dropwise 3.0 ml (25 mmol) of benzyl bromide, followedby stirring at room temperature for 24 hours. After addition of 300 mlof 0.1 N HCl, the resulting mixture was extracted twice with 200 mlportions of ethyl acetate. The ethyl acetate layer was washed with asaturated aqueous solution of sodium chloride, and then dried overanhydrous sodium sulfate. After the solvent was removed under reducedpressure, the residue was subjected to purification using 300 ml ofsilica gel (Wako Gel C-200), and as the developer, a hexane:ethylacetate mixture (3:1). The solvent was removed from the desiredfractions under reduced pressure to give 4.7 g (17 mmol) of Compound (2)(yield: 100%).

In 70 ml of methanol was dissolved 11.8 g (103 mmol) of methylazidoacetate in a stream of argon, and 20.9 ml (103 mmol) of 28% sodiummethoxide was added dropwise thereto at -50° C. over 75 minutes,followed by stirring for 30 minutes. To the resulting solution was added40 ml of a solution of 4.7 g of Compound (2) in a methanol-toluenemixture (1:1) over 25 minutes, and the temperature of the mixture wasallowed to rise from -50° C. to about -10° C. with stirring for 24hours. Then, appropriate amounts of water and diethyl ether were addedthereto to extract the ether layer. The ether layer was washed with asaturated aqueous solution of sodium chloride and dried over anhydroussodium sulfate, and the solvent was removed under reduced pressure togive 2.8 g (7.6 mmol) of Compound (3) (yield: 45%).

In 750 ml of xylene was dissolved 2.8 g of Compound (3), and thesolution was heated at 140 to 150° C. for 2 hours. After the solutionwas cooled to room temperature, the solvent was removed under reducedpressure. The residue was subjected to purification using 150 ml ofsilica gel (Wako Gel C-200), and as the developer, a hexane-ethylacetate mixture (4:1). The solvent was removed from the desiredfractions under reduced pressure to give 2.4 g (7.0 mmol) of Compound(4) (yield: 93%).

In 116 ml of a tetrahydrofuran-methanol mixture (1:1) was dissolved 2.4g of Compound (4), and 471 mg of platinum dioxide was added thereto,followed by vigorous stirring in a stream of hydrogen for 24 hours. Tothe resulting mixture was added 200 mg of platinum dioxide, followed byfurther stirring in a stream of hydrogen for 7 hours. The catalyst wasremoved by filtration using Celite, and the solvent was removed from thefiltrate under reduced pressure. The residue was subjected topurification using a column of silica gel (120 ml), and as thedeveloper, a hexane-ethyl acetate mixture (3:1). The solvent was removedfrom the desired fractions under reduced pressure to give 1.6 g (6.4mmol) of Compound (5) (yield: 91%).

In 5 ml of DMF was dissolved 100 mg (0.4 mmol) of Compound (5), and 275mg (2 mmol) of anhydrous potassium carbonate was added thereto. To theresulting solution was added dropwise 173 μl (2 mmol) of1,2-dibromoethane, followed by stirring in a stream of nitrogen at roomtemperature for 19 hours. After addition of a phosphate buffer (pH 7),the mixture was extracted with ethyl acetate. The ethyl acetate layerwas washed with a saturated aqueous solution of sodium chloride, andthen dried over anhydrous sodium sulfate. After the solvent was removedunder reduced pressure, the residue was subjected to purification using20 ml of silica gel (Wako Gel C-200), and as the developer, ahexane-ethyl acetate mixture (2:1). The solvent was removed from thedesired fractions under reduced pressure to give 94 mg (0.26 mmol) ofCompound (6) (yield: 65%).

In 9.5 ml of DMF was dissolved 94 mg (0.26 mmol) of Compound (6), and 85mg (1.3 mmol) of sodium azide was added thereto, followed by stirring atroom temperature for 25 hours. To the resulting solution were addedappropriate amounts of ethyl acetate and a phosphate buffer (pH 7) toextract the ethyl acetate layer. After the ethyl acetate layer was driedover anhydrous sodium sulfate, the solvent was removed under reducedpressure to give 79 mg of Compound (7) (yield: 95%).

In a mixture of THF (8 ml) and water (10 ml) was dissolved 79 mg ofCompound (7), and 2.5 ml of 1N aqueous solution of sodium hydroxide wasadded thereto, followed by stirring at room temperature for 3.5 hours.The reaction mixture was made acidic by addition of 1N HCl, andextracted using chloroform. The chloroform layer was washed with asaturated aqueous solution of sodium chloride and dried over anhydroussodium sulfate, and the solvent was removed under reduced pressure togive 75 mg of Compound (8) (yield: 98%).

In 7 ml of methylene chloride was dissolved 75 mg of Compound (8) underice cooling, and 103 mg (0.5 mmol) of DCC was added thereto, followed bystirring under ice cooling for one hour. To the resulting solution wereadded 70 mg (0.5 mmol) of p-nitrophenol and 61 mg (0.5 mmol) ofdimethylaminopyridine, followed by stirring at a temperature of 0° C. toroom temperature for 80 minutes. The insoluble matter was removed byfiltration, and 0.5 N HCl was added to the filtrate, followed byextraction with chloroform. The chloroform layer was washed with asaturated aqueous solution of sodium hydrogencarbonate and a saturatedaqueous solution of sodium chloride, and then dried over anhydroussodium sulfate. After the solvent was removed under reduced pressure,the product was recrystallized from ethanol to give 72 mg of Compound(9) (yield: 69%).

In a stream of argon, 37 mg (0.9 mmol) of 50% sodium hydride wasdissolved in 1.5 ml of DMF, and a solution prepared by dissolving 185 mg(0.7 mmol) of Compound (10) obtained according to the method describedin Japanese Published Unexamined Patent Application No. 178858/93 in 2.9ml of DMF was added thereto at -20° C., followed by stirring for 3hours. To the resulting solution was added a solution prepared bydissolving 368 mg (0.9 mmol) of Compound (9) in 6 ml of DMF, and thetemperature of the mixture was allowed to rise from -20° C. to roomtemperature with stirring for 24 hours. To the resulting mixture wereadded appropriate amounts of ethyl acetate and a phosphate buffer (pH7.0) to extract the ethyl acetate layer. The ethyl acetate layer waswashed with a saturated aqueous solution of sodium chloride, and driedover anhydrous sodium sulfate. After the solvent was removed underreduced pressure, the residue was subjected to purification using 80 mlof silica gel, and as the developer, a chloroform-methanol mixture(100:1). The solvent was removed from the desired fractions underreduced pressure to give 278 mg (0.5 mmol) of Compound (11) (yield:71%).

The structures of Compounds (2) to (9) and Compound (11) were confirmedby ¹ H-NMR and mass spectrometric analysis.

In a stream of nitrogen, 20 mg (36.6 μmol) of Compound (11) wasdissolved in 1.1 ml of a mixture of acetic acid (0.2 ml) andtetrahydrofuran (9.8 ml), and 7.3 mg of 10% palladium carbon catalystwas added thereto at 10° C. to 15° C., followed by vigorous stirring ina stream of hydrogen at 10° C. to 15° C. for 3 hours and 20 minutes.After the mixture was cooled to a temperature below -20° C. and thecatalyst was removed by filtration, the solvent was removed underreduced pressure under cooling to give 9 mg (17.3 μmol) of Compound (12)(yield: 47%).

¹ HNMR spectrum (500 MHz, in CDCl₃) δ (ppm): 11.58 (1H, brs, 1-NH), 9.40(1H, brs, 1'-NH), 7.12 (1H, s, H-7), 6.95 (1H, d, J=2.3 Hz, H-3'), 6.81(1H, s, H-4'), 4.45 (2H, m, H-5), 4.08 (3H, s, 7'-OCH₃), 3.92 (2H, t,J=5.2 Hz, OCH₂), 3.90 (3H, s, 5'-OCH₃), 3.82 (3H, s, 3-COOCH₃), 3.67(1H, m, H-4a), 3.20 (2H, q, J=6.4 Hz, CH₂), 2.63 (3H, s, 2-CH₃), 2.38(1H, dd, J=7.5, 3.4 Hz, H-4), 1.37 (1H, t, J=4.2 Hz, H-4); Mass spectrum(SIMS): 521 (M+H)

REFERENCE EXAMPLE 16

Compound (X-1): BzlO-PEG-Ala-Val-Compound (12)

In 2.4 ml of methylene chloride was dissolved 13 mg (15.2 μmol) ofCompound (VIII-1) obtained in Reference Example 1, and 2.0 mg of HONSuand 4.3 mg of DCC were successively added thereto under ice cooling,followed by stirring for 2 hours. The insoluble matter was removed byfiltration, and the solvent was removed from the filtrate under reducedpressure. Then the residue was dissolved in 2 ml of pyridine, followedby addition of a solution prepared by dissolving 7.8 mg (15.0 μmol) ofCompound (12) obtained in Example 24 in 1.5 ml of pyridine under icecooling. The resulting mixture was stirred under ice cooling for onehour, and then at room temperature for 2 hours. After the solvent wasremoved under reduced pressure, the residue was subjected topurification using 5 ml of silica gel (Wako Gel C-200), and as thedeveloper, 5 ml each of chloroform-methanol mixtures (100:1, 80:1, 60:1,40:1, 20:1, 10:1, 5:1). The eluate was taken in 0.5 ml fractions. Thesolvent was removed from the desired fractions under reduced pressure togive 2.8 mg (0.2 μmol) of BzlO-PEG-Ala-Val-Compound (12) (yield: 1.4%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=10:1; Rf value: 0.5; ¹ HNMR spectrum (500 MHz, inCDCl₃) δ (ppm): BzlO-PEG-Ala-Val moiety 7.36 (5H, m, C₆ H₅), 5.19 (2H,s, CH₂), 4.12 (4H, s, OCH₂), 3.64 (4nH, brs, OCH₂ CH₂), 3.23 [1H, s,CH(Ala)], 2.23 [1H, brq, J=6.0 Hz, CH(Val)], 1.26 [1H, s, CH(Val)], 1.17[3H, d, J=2.8 Hz, CH₃ (Ala)], 0.89 [6H, q, J=2.5 Hz, CH₃ (Val)],Compound (12) moiety 11.58 (1H, brs, 1-NH), 9.40 (1H, brs, 1'-NH), 7.12(1H, s, H-7), 6.95 (1H, d, J=2.3 Hz, H-3'), 6.81 (1H, s, H-4'), 4.45(2H, m, H-5), 4.08 (3H, s, 7'-OCH₃), 3.90 (3H, s, 5'-OCH₃), 3.88 (2H, t,J=5.2 Hz, OCH₂), 3.82 (3H, s, 3-COOCH₃), 3.67 (1H, m, H-4a), 3.20 (2H,q, J=6.4 Hz, CH₂), 2.63 (3H, s, 2-CH₃), 2.38 (1H, dd, J=7.5, 3.4 Hz,H-4), 1.37 (1H, t, J=4.2 Hz, H-4); Infrared absorption spectrum (inchloroform): 3595, 3450, 3010, 2900, 1750, 1665, 1608, 1515, 1460, 1310,1110 cm⁻¹

REFERENCE EXAMPLE 17

Compound (20)

Compound (20) was synthesized according to the following reaction steps.##STR23##

In a mixture of methanol (30 ml) and benzene (105 ml) was dissolved 1.51g (7.78 mmol) of 3'-hydroxy-4'-methoxycinnamic acid [Compound (13)], and11 ml (9.6 mmol) of a 10% solution of trimethylsilyl diazomethane inhexane was added thereto, followed by stirring at room temperature for 3hours. To the resulting solution was added 2.0 ml of a 10% solution oftrimethylsilyl diazomethane in hexane, followed by stirring for onehour. After addition of a saturated aqueous solution of sodiumhydrogencarbonate, the reaction mixture was extracted with ethylacetate. The ethyl acetate layer was washed with a saturated aqueoussolution of sodium chloride and dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure to give 1.55 g of methylester of 3'-hydroxy-4'-methoxycinnamic acid [Compound (14)] (yield:95.6%).

In 60 ml of DMF was dissolved 1.55 g (7.44 mmol) of Compound (14), and5.14 g (37.2 mmol) of potassium carbonate and 3.78 ml (37.2 mmol) of1,3-dibromopropane were added thereto, followed by stirring at roomtemperature for 14 hours. To the resulting solution were added water andethyl acetate to extract the ethyl acetate layer. The ethyl acetatelayer was washed with water and a saturated aqueous solution of sodiumchloride, and then dried over anhydrous sodium sulfate. After thesolvent was removed under reduced pressure, the residue was subjected topurification by silica gel chromatography using a hexane-ethyl acetatemixture (4:1) as the developer to give 1.90 g of methyl ester of3'-(3-bromopropyloxy)-4'-methoxycinnamic acid [Compound (15)] (yield:77.6%).

In 120 ml of DMF was dissolved 1.90 g (5.77 mmol) of Compound (15), and1.88 g (28.9 mmol) of sodium azide was added thereto, followed bystirring at room temperature for 17 hours. To the resulting solution wasadded a saturated aqueous solution of sodium hydrogencarbonate, followedby extraction with ethyl acetate. The ethyl acetate layer was washedwith water and a saturated aqueous solution of sodium chloride, and thendried over anhydrous sodium sulfate. The solvent was removed underreduced pressure to give 1.81 g of crude methyl ester of3'-(3-azidopropyloxy)-4'-methoxycinnamic acid [Compound (16)].

¹ HNMR spectrum (100 MHz, in CDCl₃) δ (ppm): 7.62 (1H, d, J=15.8 Hz),7.12 (1H, d, J=7.9 Hz), 7.08 (1H, s), 6.86 (1H, d, J=7.9 Hz), 6.29 (1H,d, J=15.8 Hz), 4.12 (2H, t, J=6.2 Hz), 3.89 (3H, s), 3.79 (3H, s), 3.55(2H, t, J=6.6 Hz), 2.10 (2H, m)

In a mixture of THF (60 ml) and water (2 ml) was dissolved 1.81 g ofcrude Compound (16), and 11.5 ml of 1N aqueous solution of sodiumhydroxide was added thereto, followed by stirring at room temperaturefor 19 hours. After the reaction mixture was adjusted to pH 4 byaddition of 2N HCl, water and ethyl acetate were added thereto toextract the ethyl acetate layer. The ethyl acetate layer was washed withwater and a saturated aqueous solution of sodium chloride, and thendried over anhydrous sodium sulfate. The solvent was removed underreduced pressure to give 1.64 g of crude3'-(3-azidopropyloxy)-4'-methoxycinnamic acid [Compound (17)].

In 150 ml of methylene chloride was dissolved 5.77 mmol of Compound(17), and 1.36 g (9.8 mmol) of 4-nitrophenol, 2.73 ml (19.6 mmol) oftriethylamine and 2.51 g (9.8 mmol) of 2-chloro-1-methylpyridiniumiodide were added thereto, followed by stirring at room temperature for5 hours. To the resulting solution were further added 722 mg (5.2 mmol)of 4-nitrophenol, 1.45 ml (10.4 mmol) of triethylamine and 1.33 g (5.2mmol) of 2-chloro-1-methylpyridinium iodide, followed by stirring for 17hours. After addition of a saturated aqueous solution of sodiumhydrogencarbonate, the reaction mixture was extracted with chloroform.The chloroform layer was washed with a saturated aqueous solution ofsodium hydrogencarbonate and dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure. The residue wassubjected to purification by silica gel chromatography using, as thedeveloper, hexane-ethyl acetate mixtures (4:1-2:1) to give 2.15 g of4-nitrophenyl ester of 3'-(3-azidopropyloxy)-4'-methoxycinnamic acid[Compound (18)]. The obtained compound was recrystallized from ethanolto give 2.01 g of Compound (18) (yield: 87%).

¹ HNMR spectrum (100 MHz, in CDCl₃) δ (ppm): 8.30 (1H, d, J=9.2 Hz),8.17 (2H, d, J=9.2 Hz), 7.84 (1H, d, J=15.8 Hz), 7.37 (1H, d, J=9.0 Hz),7.16 (1H, s), 6.90 (2H, d, J=9.2 Hz), 6.47 (1H, d, J=15.8 Hz), 4.15 (1H,t, J=6.2 Hz), 3.92 (3H, s), 3.57 (2H, t, J=6.5 Hz), 2.12 (2H, m)

To 12 mg (0.3 mmol) of 60% sodium hydride were added 0.6 ml of DMF, andthen 1.5 ml of a solution of 60 mg (0.23 mmol) of Compound (10) in DMF,followed by stirring in an atmosphere of argon at 0° C. for 2 hours.After the resulting mixture was cooled to -20° C., 1.5 ml of a solutionof 124 mg (0.31 mmol) of Compound (18) in DMF was added thereto,followed by stirring at -20 to 0° C. for 2 hours. To the resultingmixture was added 0.2 M phosphate buffer (pH 7), and then the mixturewas extracted with ethyl acetate. The ethyl acetate layer was washedwith a saturated aqueous solution of sodium chloride and dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure. The residue was subjected to purification using 30 ml ofsilica gel, and as the developer, a chloroform-methanol mixture (50:1)to give 77 mg of Compound (19) (yield: 65%).

¹ HNMR spectrum (270 MHz, in CDCl₃) δ (ppm): 9.81 (1H, br), 7.68 (1H, d,J=15.5 Hz), 7.11 (1H, dd, J=8.3, 2.0 Hz), 7.01 (1H, d, J=2.0 Hz), 6.81(1H, d, J=8.2 Hz), 6.66 (1H, d, J=15.5 Hz), 6.56 (1H, br), 4.15 (1H, d,J=11.2 Hz), 4.07 (2H, t, J=3.6 Hz), 4.06 (1H, m), 3.84 (3H, s), 3.76(3H, s), 3.50 (2H, t, J=6.6 Hz), 3.46 (1H, m), 2.52 (3H, s), 2.31 (1H,dd, J=7.3, 3.3 Hz), 2.05 (2H, m), 1.25 (1H, dd, J=5.3, 3.4 Hz); Infraredabsorption spectrum (KBr): 2098, 1697, 1622, 1608, 1516, 1392, 1263,1217 cm⁻¹ ; Mass spectrum (SIMS): 518 (M+H)

In 1.5 ml of THF was dissolved 15 mg (0.029 mmol) of Compound (19), and23 mg (0.088 mmol) of triphenylphosphine was added thereto, followed bystirring at room temperature for 30 minutes. To the resulting solutionwas added 1.5 ml of water, followed by stirring at room temperature for24 hours. After addition of a saturated aqueous solution of sodiumhydrogencarbonate, the reaction mixture was extracted with chloroform.The chloroform layer was washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate, and the solventwas removed under reduced pressure. The residue was subjected topurification using 30 ml of silica gel, and as the developer, achloroform-methanol-triethylamine mixture (200:10:1) to give 4 mg ofCompound (20) (yield: 28%).

¹ HNMR spectrum (270 MHz, in DMSO-d₆) δ (ppm): 7.75 (1H, d, J=15.2 Hz),7.54 (1H, brs), 7.49 (1H, br, J=8.6 Hz), 7.18 (1H, d, J=8.6 Hz), 7.09(1H, d, J=15.2 Hz), 7.03 (1H, br), 4.15 (1H, brd, J=11.2 Hz), 4.39 (1H,m), 4.27 (2H, t, J=3.6 Hz), 3.96 (3H, s), 3.87 (3H, s) 3.61 (1H, m),3.12 (2H, t, J=7.2 Hz), 2.61 (3H, s), 2.23 (1H, m), 2.16 (2H, m), 1.46(1H, m); Infrared absorption spectrum (KBr): 1647, 1610, 1512, 1458,1394, 1385, 1294, 1219 cm⁻¹ ; Mass spectrum (SIMS): 492 (M+H)

REFERENCE EXAMPLE 18

Compound (X-2): BzlO-PEG-Ala-Pro-Compound (12)

In 3.5 ml of methylene chloride was dissolved 21 mg (24.5 μmol) ofCompound (VIII-2) obtained in Reference Example 2, and 3.4 mg of HONSuand 6.3 mg of DCC were successively added thereto under ice cooling,followed by stirring for 2 hours. After the insoluble matter was removedby filtration, the solvent was removed from the filtrate under reducedpressure. The residue was dissolved in 3.4 ml of pyridine, followed byaddition of a solution of 9.9 mg (19 μmol) of Compound (12) obtained inReference Example 15 in 2.0 ml of pyridine under ice cooling. Theresulting mixture was stirred under ice cooling for one hour and then atroom temperature for 2 hours. After the solvent was removed underreduced pressure, the residue was subjected to purification using 10 mlof silica gel (Wako Gel C-200), and as the developer, 10 ml each ofchloroform-methanol mixtures (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1).The eluate was taken in 1.0 ml fractions. The solvent was removed fromthe desired fractions under reduced pressure to give 10.0 mg (7.3 μmol)of BzlO-PEG-Ala-Pro-Compound (12) (yield: 39%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=10:1; Rf value: 0.5; ¹ HNMR spectrum (500 MHz, inCDCl₃) δ (ppm): BzlO-PEG-Ala-Pro moiety 7.36 (5H, m, C₆ H₅), 5.19 (2H,s, CH₂), 4.50 [2H, brm, CH₂ (Pro)], 4.12 (4H, s, OCH₂), 3.64 (4nH, brs,OCH₂ CH₂), 3.42 [2H, br, CH₂ (Pro)], 3.23 [1H, s, CH(Ala)], 2.37 [1H,br, CH(Pro)], 2.08 [2H, brm, CH₂ (Pro)], 1.17 [3H, d, J=2.8 Hz, CH₃(Ala)], Compound (12) moiety 11.58 (1H, brs, 1-NH), 9.40 (1H, brs,1'-NH), 7.12 (1H, s, H-7), 6.95 (1H, d, J=2.3 Hz, H-3'), 6.81 (1H, s,H-4'), 4.45 (2H, m, H-5), 4.08 (3H, s, 7'-OCH₃), 3.92 (2H, t, J=5.2 Hz,OCH₂), 3.90 (3H, s, 5'-OCH₃), 3.82 (3H, s, 3-COOCH₃), 3.67 (1H, m,H-4a), 3.20 (2H, q, J=6.4 Hz, CH₂), 2.63 (3H, s, 2-CH₃), 2.38 (1H, dd,J=7.5, 3.4 Hz, H-4), 1.37 (1H, t, J=4.2 Hz, H-4); Infrared absorptionspectrum (in chloroform): 3595, 3450, 3010, 2900, 1750, 1665, 1608,1515, 1460, 1310, 1110 cm⁻¹

REFERENCE EXAMPLE 19

Compound (25)

Compound (25) was synthesized according to the following reaction steps.##STR24##

In 75 ml of methylene chloride was dissolved 3.1 g (15 mmol) of2-bromoethylamine hydrobromide, and the solution was cooled to -40 to-50° C. After 2.6 ml (18 mmol) of benzyloxycarbonyl chloride was addeddropwise thereto, the temperature of the mixture was allowed to rise toroom temperature over 24 hours. Then, the solvent was removed underreduced pressure, and 50 ml of ethyl acetate and 50 ml of 1N HCl wereadded to the residue, followed by stirring at room temperature for 6hours. The ethyl acetate layer was washed with 50 ml of a saturatedaqueous solution of sodium chloride and dried over anhydrous sodiumsulfate, and the solvent was removed under reduced pressure to give 2.0g of Compound (21) (yield: 53%).

In 10 ml of DMF was dissolved 495 mg (2.0 mmol) of Compound (5), and 545mg of anhydrous potassium carbonate and 1020 mg (4.0 mmol) of Compound(21) were successively added thereto, followed by stirring under icecooling for 24 hours. To the resulting solution were successively added100 ml of a phosphate buffer (20 mmol, pH 7.0) and 100 ml of ethylacetate to extract the ethyl acetate layer. The ethyl acetate layer waswashed with a saturated aqueous solution of sodium chloride and driedover anhydrous sodium sulfate, and the solvent was removed under reducedpressure. The residue was subjected to purification using 100 ml ofsilica gel (Wako Gel C-200), and as the developer, 200 ml each ofhexane-ethyl acetate mixtures (5:1, 4:1, 3:1). The eluate was taken in10 ml fractions. The solvent was removed from the desired fractionsunder reduced pressure, the desired fractions being identified by silicagel thin layer chromatography [Kieselgel 60 (Merck & Co., Inc.),hexane:ethyl acetate=2:1, Rf value: 0.2], whereby 713 mg (1.7 mmol) ofCompound (22) was obtained (yield: 83%).

To 66 mg (0.15 mmol) of Compound (22) were added 1.5 ml of THF and 1.5ml of methanol, and 6.6 mg of 10% palladium carbon catalyst was addedthereto, followed by vigorous stirring in a stream of hydrogen for 18hours. To the resulting mixture was added 6.6 mg of the catalyst,followed by further stirring for 5 hours. After the catalyst was removedby filtration, the solvent was removed from the filtrate under reducedpressure to give 36 mg (0.12 mmol) of Compound (23) (Segment B) (yield:82%).

Silica gel thin layer chromatography: Kieselgel 60Chloroform:methanol=5:1; Rf value: 0.3

In 1.5 ml of methylene chloride was dissolved 109 mg (0.13 mmol) ofCompound (VIII-1) obtained in Reference Example 1, and 21 mg of HONSuand 38 mg of DCC were successively added thereto under ice cooling,followed by stirring under ice cooling for 3 hours. After the insolublematter (DCU) was removed by filtration, the solvent was removed from thefiltrate under reduced pressure. To the residue was added 1 ml ofmethylene chloride, and 1 ml of a solution of 34 mg (0.12 mmol) ofCompound (23) in methylene chloride was added thereto under ice cooling.The temperature of the mixture was allowed to rise to room temperaturewith stirring for 24 hours. After the insoluble matter (DCU) was removedby filtration, the solvent was removed from the filtrate under reducedpressure to obtain 192 mg of a residue. The residue was subjected topurification using 20 ml of silica gel (Wako Gel C-200), and as thedeveloper, 20 ml each of chloroform-methanol mixtures (100:0, 100:1,50:1, 30:1, 20:1). The eluate was taken in 5 ml fractions. The solventwas removed from the desired fractions under reduced pressure, thedesired fractions being identified by silica gel thin layerchromatography [Kieselgel 60 (Merck & Co., Inc.),chloroform:methanol=10:1, Rf value: 0.4], whereby 89 mg (0.08 mmol) ofCompound (24) was obtained (yield: 68%). The structures of Compounds(21) to (23) were confirmed by ¹ HNMR and mass spectrometric analysis,and the structure of Compound (24) was confirmed by ¹ HNMR.

In a mixture of 2 ml of THF and 2 ml of methanol was dissolved 89 mg (79μmol) of Compound (24), and 18 mg of 10% palladium carbon catalyst wasadded thereto, followed by vigorous stirring in a stream of hydrogen for15 hours. After addition of 9 mg of the catalyst, the mixture wasstirred in a stream of hydrogen for 9 hours. Then, 9 mg of the catalystwas added again, followed by stirring in a stream of hydrogen for 15hours. After the catalyst was removed by filtration, the solvent wasremoved from the filtrate under reduced pressure. The residue wassubjected to purification using 10 ml of silica gel (Wako Gel C-200),and as the developer, chloroform-methanol mixtures (100:0, 50:1, 30:1).The eluate was taken in 5 ml fractions. The solvent was removed from thedesired fractions under reduced pressure, the desired fractions beingidentified silica gel thin layer chromatography, whereby 52 mg ofCompound (25) was obtained (yield: 63%).

Silica gel thin layer chromatography: Kieselgel 60 (Merck & Co., Inc.);Chloroform:methanol=5:1; Rf value: 0.4; ¹ HNMR spectrum (500 MHz, inCDCl₃) δ (ppm): PEG-Ala-Val moiety 4.13 (4H, s, OCH₂), 3.64 (4nH, brs,OCH₂ CH₂), 3.23 [1H, s, CH(Ala)], 2.22 [1H, brq, J=6.0 Hz, CH(Val)],1.26 [1H, s, CH(Val)], 1.17 [3H, d, J=2.8 Hz, CH₃ (Ala)], 0.89 [6H, q,J=2.5 Hz, CH₃ (Val)], Segment B moiety 9.40 (1H, brs, 1-NH), 6.95 (1H,d, J=2.2 Hz, H-3), 6.82 (1H, s, H-4), 4.08 (3H, s, 7-OCH₃), 3.92 (2H, d,J=5.2 Hz, OCH₂), 3.90 (3H, s, 5-OCH₃), 3.85 (3H, s, 2-COOCH₃), 3.20 (2H,q, J=6.4 Hz, CH₂)

REFERENCE EXAMPLE 20

KM-641-(PEG-Ala-Val-Segment B)_(m)

In 0.8 ml of methylene chloride was dissolved 7.8 mg (7.5 μmol) ofCompound (25) obtained in Reference Example 19, and 1.0 ml of a solutionof HONSu in methylene chloride (1 mg/ml) and 0.9 ml of a solution of DCCin methylene chloride (2 mg/ml) were successively added thereto underice cooling, followed by stirring at room temperature for 3 hours. Afterthe solvent was removed under reduced pressure, 1 ml of DMSO was addedto the residue. The resulting mixture was added under ice cooling to asolution prepared by adding 11.5 ml of a phosphate buffer to 8 ml of anaqueous solution of KM-641 antibody (1.47 mg/ml), followed by gentlestirring at 4° C. for 24 hours. After the insoluble matter was removedwith a filter (0.22 μm), the antibody fraction was purified by gelfiltration chromatography [column: 200 ml of Sephacryl S 200 (PharmaciaCo., Ltd.), developer: a phosphate buffer, flow rate: 0.5 ml/minute,11.5 ml fractions]. The 8th and 9th fractions were collected to obtain asolution containing 0.34 mg/ml KM-641-(PEG-Ala-Val-Segment B)_(m).

The number of molecules of Segment B bound per antibody molecule wascalculated by subjecting the conjugate to enzyme treatment (thermolysin)and quantitatively determining released H-Val-Segment B by HPLCaccording to the method described in Reference Example 22. It was foundthat in the obtained conjugate, the number of molecules of Segment B was1.9 per antibody molecule.

It was confirmed that the affinity of the conjugate was approximatelyequal to that of an unbound antibody according to the followingenzyme-linked immunosorbent assay.

<Measurement of an affinity of an antibody by ELISA (Enzyme-linkedImmunosorbent Assay)>

Ganglioside GD₃ (2 nmol) was dissolved in 2 ml of ethanol containing 5ng of phosphatidyl choline (Sigma Chemical Co.) and 2.5 ng ofcholesterol, and the solution was put into wells of a 96-well plate forELISA (Linbro Co., Ltd.) in an amount of 20 μl/well. After drying thewells, a phosphate buffer containing 1% bovine serum albumin was addedto the wells for blocking. The above-described conjugate (10 μg/ml, 50μl) was added to each well, and the plate was allowed to stand at roomtemperature for 2 hours (or at 4° C. for 24 hours). Thenperoxidase-labelled rabbit anti-mouse Ig antibody (Dako) was added tothe wells as the second antibody, and the plate was allowed to stand atroom temperature for 1 to 2 hours, followed by washing. ABTS (SigmaChemical Co.) solution was added, and after the color developed,spectrophotometry was carried out on the absorbance at 414 nm usingNJ-2001 (Japan Intermed Co., Ltd.).

REFERENCE EXAMPLE 21

Enzyme-specific Cleavage of a Spacer Experiment using a spacer bound toSegment B (side chain model)

This Reference Example demonstrates that the peptide bond of a spacerbound to an antitumor agent is cleaved in a cell by a specific enzymeand that the cleavage of the spacer does not occur in a serum, usingCompound (25) obtained in Reference Example 19. The release of Segment Bby the use of thermolysin as the cleavage enzyme was confirmed in thefollowing manner.

To 0.1 ml of a solution of Compound (25) in a phosphate buffer (0.2mg/ml) was added 0.1 ml of an enzyme solution (0.1 mg/ml) (amount ofenzyme: 94 pU), and the mixture was allowed to stand at 37° C. for 24hours. The amount of H-Val-Segment B released from Compound (25) waschecked by analyzing the supernatant by reversed-phase HPLC (cleavageefficiency: 100%).

Reversed-phase HPLC conditions;

Apparatus: UVIDEC-100IV Spectrophotometric detector, TRIROTAR SR (JapanSpectroscopic Co., Ltd.); Column: UNISIL PACK 5C18-150A (GL Sciences);Eluent: 50 mM acetate buffer (pH 4.5) [10-70% acetonitrile gradient (35minutes)]; Flow rate: 0.7 ml/minute; Detection wavelength: 300 nm;Elution time: 39.0 minutes; Mass spectrum (SIMS): 393.2 (M+H); Aminoacid analysis: Val 1.0 (1.0)

It was confirmed as follows that the spacer was not cleaved in a serum.

To 0.1 ml of a solution of Compound (25) in a phosphate buffer (0.2mg/ml) was added 0.1 ml of a human serum, and the mixture was allowed tostand at 37° C. for 2 days. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in the aboveexperiment, except that 50 mM phosphate buffer (pH 5.9) was used as theeluent. As a result, the peak derived from Segment B was not confirmed.

REFERENCE EXAMPLE 22

Enzyme-specific Cleavage of a Spacer Experiment using a conjugate of anantibody and Segment B through a spacer (side chain model)

This Reference Example demonstrates that the peptide bond of a conjugateof an antibody and an antitumor agent through a spacer is cleaved in acell by a specific enzyme, but is stable in a serum, using the conjugateof an antibody and Segment B through a spacer obtained in ReferenceExample 20. The specific cleavage of the spacer was confirmed as followsusing thermolysin as the intracellular cleavage enzyme and plasmin asthe main proteolytic enzyme in blood.

To 250 μl of KM-641-(PEG-Ala-Val-Segment B)_(m) (0.33 mg/ml) were added2.5 μl of thermolysin (0.1 mg/ml) (amount of enzyme: 2.4 pU) [or 2.5 μlof a 1:200 dilution of plasmin (amount of enzyme: 250 μU)] and 5.7 μl ofa phosphate buffer, and the mixture was allowed to stand at 37° C. for24 hours. The resulting supernatant was analyzed by reversed-phase HPLCunder the same conditions as in Reference Example 21.

The peak of H-Val-Segment B similar to that in Reference Example 21 wasconfirmed 42.0 minutes after the elution started for the conjugatetreated with thermolysin, but the peak was not confirmed for theconjugate treated with plasmin. Plasmin being one of the mainproteolytic enzymes in blood, these results show that the conjugate isstable in blood, but when it is incorporated into a cell, its antitumoragent moiety is specifically cleaved by a specific enzyme to express anantitumor activity.

REFERENCE EXAMPLE 23

Enzyme-specific Cleavage of a Spacer Experiment using a spacer bound toCompound (12)

(1) Cleavage of Compound (X-1) [BzlO-PEG-Ala-Val-Compound (12)] withthermolysin

In 10 μl of DMSO was dissolved 20 μg (15 nmol) of Compound (X-1)obtained in Reference Example 16, and 90 μl of a phosphate buffer wasadded thereto. To the resulting mixture was added 100 μl of athermolysin solution (2 mg/ml) (amount of enzyme: 1.9 μU), and themixture was allowed to stand at 37° C. for 5 hours. The resultingsupernatant was analyzed by reversed-phase HPLC, whereby the release ofH-Val-Compound (12) from Compound (X-1) (elution time: 35.1 minutes) wasconfirmed (cleavage efficiency: 78%).

Reversed-phase HPLC conditions; Apparatus: The same as in ReferenceExample 21; Eluent: A solution containing 25% buffer (pH 4.8) preparedfrom a 0.2 M aqueous solution of disodium hydrogenphosphate and a 0.1 Maqueous solution of citric acid [10-70%. acetonitrile gradient (35minutes)]; Flow rate: 0.7 ml/minute; Detection wavelength: 330 nm;Elution time: 31.6 minutes; Mass spectrum (SIMS): 620 (M+H); Amino acidanalysis: Val 1.0 (1.0)

(2) Cleavage of Compound (X-2) [BzlO-PEG-Ala-Pro-Compound (12)] withproline endopeptidase

To 54 μg (40 nmol) of Compound (X-2) obtained in Reference Example 18were added 20 μl of DMSO and 370 μl of a phosphate buffer. After 10 μlof a proline endopeptidase solution (0.1 mg/ml) was added, the mixturewas allowed to stand at 37° C. for 2.5 hours. The resulting supernatantwas analyzed by reversed-phase HPLC, whereby the release of Compound(12) from Compound (X-2) was confirmed (cleavage efficiency: 100%).

Reversed-phase HPLC conditions;

Apparatus: The same as in Reference Example 21; Eluent: 50 mM phosphatebuffer (pH 5.9) (10-70% acetonitrile gradient); Flow rate: 0.7ml/minute; Detection wavelength: 330 nm; Elution time: 30.6 minutes[agreed with that for Compound (12)]; Mass spectrum (SIMS): 521 (M+H)

REFERENCE EXAMPLE 24

Enzyme-specific Cleavage of a Spacer Experiment using a conjugate of anantibody and Compound (12) through a spacer

To 740 μl of Compound (Ia-11) {KM-641-[PEG-Ala-Val-Compound (12)]_(m) }obtained in Example 11 (0.07 mg/ml) was added 5.1 μl of a thermolysinsolution (0.1 mg/ml) (amount of enzyme: 26 μU), and the mixture wasallowed to stand at 37° C. for 8 hours. The resulting supernatant wasanalyzed by reversed-phase HPLC under the same conditions as inReference Example 23 (1), whereby the release of H-Val-Compound (12)from Compound (Ia-11) was confirmed.

Elution time: 31.6 minutes; Mass spectrum (SIMS): 620 (M+H)

REFERENCE EXAMPLE 25

Enzyme-specific Cleavage of a Spacer Experiment using a spacer bound toadriamycin

(1) Cleavage of Compound (XI-3) (HO-PEG-Gly-Pro-ADM) with prolineendopeptidase

In 20 μl of DMSO was dissolved 14.3 μg (11 nmol) of Compound (XI-3)obtained in Reference Example 8, and 210 μl of a phosphate buffer wasadded thereto. To the resulting mixture was added 80 μl of prolineendopeptidase (1.0 mg/ml) (amount of enzyme: 2.8 U), and the mixture wasallowed to stand at 37° C. for 24 hours. The resulting supernatant wasanalyzed by reversed-phase HPLC, whereby the release of ADM fromCompound (XI-3) (elution time: 23.3 minutes) was confirmed (cleavageefficiency: 50%).

Reversed-phase HPLC conditions; Eluent: 50 mM phosphate buffer (pH 5.9)[10-70% acetonitrile gradient (35 minutes)]; Flow rate: 0.7 ml/minute;Detection wavelength: 233 nm; Elution time: 24.0 minutes (agreed withthat for ADM); Mass spectrum (SIMS): 544.2 (M+H) (agreed with that forADM)

(2) Cleavage of Compound (XI-1) (HO-PEG-Ala-Val-ADM) with thermolysin

In 10 μl of DMSO was dissolved 0.09 mg (10.2 nmol) of Compound (XI-1)obtained in Example 6, and 210 μl of a phosphate buffer was addedthereto. To the resulting mixture was added 80 μl of thermolysin (1.0mg/ml) (amount of enzyme: 750 pU), and the mixture was allowed to standat 37° C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in (1), whereby therelease of H-Val-ADM from Compound (XI-1) (elution time: 23.8 minutes)was confirmed (cleavage efficiency: 100%).

Elution time: 25.2 minutes; Mass spectrum (SIMS): 643 (M+H)

REFERENCE EXAMPLE 26

Enzyme-specific Cleavage of a Spacer Experiment using a conjugate of anantibody and adriamycin through a spacer

(1) Cleavage of Compound (Ia-3) [NL-1-(PEG-Gly-Pro-ADM)_(m) ] withproline endopeptidase

To 35 μl of Compound (Ia-3) obtained in Example 3 (0.19 mg/ml) wereadded 70 μl of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.4U) and 95 μl of a phosphate buffer, and the mixture was allowed to standat 37° C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in Reference Example25, whereby the release of ADM from Compound (Ia-3) was confirmed(cleavage efficiency: 10%).

Elution time: 23.7 minutes (agreed with that for ADM)

(2) Cleavage of Compound (Ia-1) [NL-1-(PEG-Ala-Val-ADM)_(m) ] withthermolysin

To 50 μl of Compound (Ia-1) obtained in Example 1 (0.42 mg/ml) wereadded 50 μl of thermolysin (2.0 mg/ml) (amount of enzyme: 0.9 μU) and100 μl of a phosphate buffer, and the mixture was allowed to stand at37° C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in Reference Example25, whereby the release of H-Val-ADM from Compound (Ia-1) was confirmed(cleavage efficiency: 100%).

Elution time: 28.1 minutes

(3) Cleavage of Compound (Ia-2) [NL-1-(PEG-Ala-Pro-ADN)_(m) ] withproline endopeptidase

To 40 μl of Compound (Ia-2) obtained in Example 2 (0.27 mg/ml) wereadded 110 μl of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 3.9U) and 50 μl of a phosphate buffer, and the mixture was allowed to standat 37° C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in Reference Example25, whereby the release of ADM from Compound (Ia-2) was confirmed(cleavage efficiency: 10%).

Elution time: 23.9 minutes (agreed with that for ADM)

REFERENCE EXAMPLE 27

Enzyme-specific Cleavage of a Spacer Experiment using a conjugate of anantibody and Compound (20) through a spacer

(1) Cleavage of Compound (Ia-9) {NL-1-[PEG-Gly-Pro-Compound (20)]_(m) }with proline endopeptidase

To 17 μl of Compound (Ia-9) obtained in Example 9 (1.2 mg/ml) were added80 μl of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.8 U) and53 μl of a phosphate buffer, and the mixture was allowed to stand at 37°C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC, whereby the release of Compound (20) from Compound(Ia-9) was confirmed.

Reversed-phase HPLC conditions; The same apparatus and column as inReference Example 21 were used. Eluent: 50 mM phosphate buffer (pH 7.0)[10-70% acetonitrile gradient (35 minutes)]; Flow rate: 0.7 ml/minute;Detection wavelength: 330 nm; Elution time: 26.0 minutes [agreed withthat for Compound (20)]

(2) Cleavage of Compound (Ia-7) {NL-1-[PEG-Ala-Val-Compound (20)]_(m) }with thermolysin

To 18 μl of Compound (Ia-7) obtained in Example 7 (1.1 mg/ml) were added60 μl of thermolysin (3.0 mg/ml) (amount of enzyme: 1.7 μU) and 72 μl ofa phosphate buffer, and the mixture was allowed to stand at 37° C. for24 hours. The resulting supernatant was analyzed by reversed-phase HPLCunder the same conditions as in (1), whereby the release ofH-Val-Compound (20) from Compound (Ia-7) was confirmed.

Elution time: 17.7 minutes

(3) Cleavage of Compound (Ia-8) {NL-1-[PEG-Ala-Pro-Compound (20)]_(m) }with proline endopeptidase

To 11 μl of Compound (Ia-B) obtained in Example 8 (1.8 mg/ml) were added80 μl of proline endopeptidase (1.0 mg/ml) (amount of enzyme: 2.8 U) and59 μl of a phosphate buffer, and the mixture was allowed to stand at 37°C. for 24 hours. The resulting supernatant was analyzed byreversed-phase HPLC under the same conditions as in (1), whereby therelease of Compound (20) from Compound (Ia-8) was confirmed.

Elution time: 25.9 minutes [agreed with that for Compound (20)]

REFERENCE EXAMPLE 28

Compound (IIa-1): HO-PEG-Ala-Val-OH

In 5.0 ml of methanol was dissolved 100 mg (117 μmol) of Compound(VIII-1) obtained in Reference Example 1 in a stream of nitrogen, and 20mg of 10% palladium carbon catalyst was added thereto, followed byvigorous stirring in a stream of hydrogen at room temperature for 2hours. After the catalyst was removed by filtration, the solvent wasremoved from the filtrate under reduced pressure to give 76 mg (99 μmol)of HO-PEG-Ala-Val-OH (yield: 85%).

¹ HNMR spectrum (100 MHz, in CDCl₃) δ (ppm): 4.12 (4H, s, OCH₂), 3.64(4nH, brs, OCH₂ CH₂), 3.23 [1H, s, CH(Ala)], 2.23 [1H, brq, J=6.0 Hz,CH(Val)], 1.26 [1H, s, CH(Val)], 1.17 [3H, d, J=2.8 Hz, CH₃ (Ala)], 0.89(6H, brd, J=2.5 Hz, CH₃ (Val)]

REFERENCE EXAMPLE 29

Compound (IIa-2): HO-PEG-Ala-Pro-OH

In 5.0 ml of methanol was dissolved 100 mg (117 μmol) of Compound(VIII-2) obtained in Reference Example 2 in a stream of nitrogen, and 20mg of 10% palladium carbon catalyst was added thereto, followed byvigorous stirring in a stream of hydrogen at room temperature for 3hours. After the catalyst was removed by filtration, the solvent wasremoved from the filtrate under reduced pressure to give 70 mg (91 μmol)of HO-PEG-Ala-Pro-OH (yield: 78%).

¹ HNMR spectrum (100 MHz, in CDCl₃) δ (ppm): 4.40 [2H, br, CH₂ (Pro)],4.12 (4H, s, OCH₂), 3.80 [1H, q, J=6.0 Hz, CH(Ala)], 3.64 (4nH, brs,OCH₂ CH₂), 3.59 [2H, br, CH₂ (Pro)], 2.36 [1H, br, CH(Pro)], 2.02 [2H,br, CH₂ (Pro)], 1.29 [3H, brd, J=3.5 Hz, CH₃ (Ala)]

REFERENCE EXAMPLE 30

Compound (IIa-3): HO-PEG-Gly-Pro-OH

In 5.0 ml of methanol was dissolved 100 mg (116 μmol) of Compound(VIII-3) obtained in Reference Example 3 in a stream of nitrogen, and 20mg of 10% palladium carbon catalyst was added thereto, followed byvigorous stirring in a stream of hydrogen at room temperature for 2hours. After the catalyst was removed by filtration, the solvent wasremoved from the filtrate under reduced pressure to give 76 mg (98 μmol)of HO-PEG-Gly-Pro-OH (yield: 85%).

¹ HNMR spectrum (100 MHz, in CDCl₃) δ (ppm): 4.40 [2H, br, CH₂ (Pro)],4.12 (4H, s, OCH₂), 3.82 [2H, s, CH₂ (Gly)], 3.64 (4nH, brs, OCH₂ CH₂),3.59 [2H, br, CH₂ (Pro)], 2.21 [1H, s, CH(Pro)], 2.02 [2H, br, CH₂(Pro)]

INDUSTRIAL APPLICABILITY

The present invention provides a toxin conjugate which is useful as anactive ingredient of an antitumor agent and an in vitro diagnosistechnique using the conjugate. The conjugate comprises a toxin and acompound having an affinity for a target cell, for example, an antibodyor antibody fragment which is specific to a cancer, said toxin andcompound being bound through a spacer.

What is claimed is:
 1. A toxin conjugate in which a compound having anaffinity for a target cell is bound to a toxin through a spacerconsisting essentially of polyalkylene glycol and a dipeptide.
 2. Atoxin conjugate represented by formula (I)

    Z.paren open-st.X.sup.1 --CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n OCH.sub.2 CO--R.sup.1 --R.sup.2 --W--Y.sup.1).sub.m                 (I)

wherein Z represents a compound having an affinity for a target cell andhaving a group capable of binding to X¹ ; X¹ represents CO, S or##STR25## W represents a single bond or ##STR26## Y¹ represents a toxin;R¹ and R², which may be the same or different, each represents an aminoacid residue; n represents an integer of 1-1000; and m represents aninteger of 1-100.
 3. The toxin conjugate according to claim 2, whereinthe group capable of binding to X¹ is CO, N, S or O, and Y¹ has a groupselected from N, S or O, provided that when X¹ is S, Z is bound to X¹via CO of Z, and when X¹ is a group other than S, Z is bound to X¹ viaN, S or O of Z, and Y¹ is bound to W or R² through N, S or O of Y¹. 4.The toxin conjugate according to claim 2 or 3, wherein Z is a protein ora peptide.
 5. The toxin conjugate according to claim 4, wherein theprotein is an antibody or an antibody fragment.
 6. The toxin conjugateaccording to claim 2 wherein R¹ represents an alanine residue, a leucineresidue, or a glycine residue; and R² represents a proline residue, avaline residue, or a leucine residue.
 7. The toxin conjugate accordingto claim 2 wherein Y¹ is adriamycin, daunorubicin, a duocarmycinderivative, mitomycin A, mitomycin C, ricin A, diphtheria toxin, orPseudomonas exotoxin.
 8. A toxin conjugate according to claim 2, whereinsaid compound having an affinity for a target cell has a group capableof binding to X¹ selected from the group consisting of COOH, NH, SH andOH.
 9. A toxin conjugate according to claim 1, wherein said toxinconjugate has a structure selected from the group consistingofNL-1-(PEG-Ala-Val-ADM)m NL-1-(PEG-Ala-Pro-ADM)mNL-1-(PEG-Gly-Pro-ADM)m KM-231-(PEG-Ala-Val-DNR)mKM-231-(PEG-Ala-Pro-DNR)m KM-231-(PEG-Gly-Pro-DNR)mNL-1-(PEG-Ala-Val-Compound (20))m NL-1-(PEG-Ala-Pro-Compound (20))mNL-1-(PEG-Gly-Pro-Compound (20))m KM-231-(PEG-Ala-Val-Compound 12)mKM-631-(PEG-Ala-Val-Compound 12)m,wherein NL-1, MK-231 and KM-631 aremonoclonal antibodies, and ADM is adriamycin and DNR is daunorubicin.10. A toxin conjugate according to claim 3, wherein said Y¹ is bound toR² through N, S or O, when W represents a single bond.
 11. A toxinconjugate in which a compound having an affinity for a target cell isbound to a toxin through a spacer consisting of a polyalkylene glycoland a dipeptide.